Oligonucleotide-based probes and methods for detection of microbes

ABSTRACT

The present invention relates to a rapid detection of microbial-associated nuclease activity with chemically modified nuclease (e.g., endonuclease) substrates, and probes and compositions useful in detection assays.

RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/937,359, filed Feb. 7, 2014, and to U.S. Provisional PatentApplication No. 61/992,034, filed May 12, 2014, and to U.S. ProvisionalPatent Application No. 61/980,498, filed Apr. 16, 2014, the entirety ofwhich are incorporated herein by reference.

FEDERAL GRANT SUPPORT

This invention was made with government support under AI083211,1R21AI101391-01A1 and 1R01AI106738-01 awarded by the National Institutesof Health. The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 3, 2015, isnamed 17023_143WO1_SL.txt and is 27,134 bytes in size.

BACKGROUND OF THE INVENTION

Chemical moieties that quench fluorescent light operate through avariety of mechanisms, including fluorescence resonance energy transfer(FRET) processes and ground state quenching. FRET is one of the mostcommon mechanisms of fluorescent quenching and can occur when theemission spectrum of the fluorescent donor overlaps the absorbancespectrum of the quencher and when the donor and quencher are within asufficient distance known as the Forster distance. The energy absorbedby a quencher can subsequently be released through a variety ofmechanisms depending upon the chemical nature of the quencher. Capturedenergy can be released through fluorescence or through nonfluorescentmechanisms, including charge transfer and collisional mechanisms, or acombination of such mechanisms. When a quencher releases captured energythrough nonfluorescent mechanisms FRET is simply observed as a reductionin the fluorescent emission of the fluorescent donor.

Although FRET is the most common mechanism for quenching, anycombination of molecular orientation and spectral coincidence thatresults in quenching is a useful mechanism for quenching by thecompounds of the present invention. For example, ground-state quenchingcan occur in the absence of spectral overlap if the fluorophore andquencher are sufficiently close together to form a ground state complex.

Quenching processes that rely on the interaction of two dyes as theirspatial relationship changes can be used conveniently to detect and/oridentify nucleotide sequences and other biological phenomena. As notedpreviously, the energy transfer process requires overlap between theemission spectrum of the fluorescent donor and the absorbance spectrumof the quencher. This complicates the design of probes because not allpotential quencher/donor pairs can be used. For example, the quencherBHQ-1, which maximally absorbs light in the wavelength range of about500-550 nm, can quench the fluorescent light emitted from thefluorophore fluorescein, which has a wavelength of about 520 nm. Incontrast, the quencher BHQ-3, which maximally absorbs light in thewavelength range of about 650-700 nm would be less effective atquenching the fluorescence of fluorescein but would be quite effectiveat quenching the fluorescence of the fluorophore Cy5 which fluoresces atabout 670 nm. The use of varied quenchers complicates assay developmentbecause the purification of a given probe can vary greatly depending onthe nature of the quencher attached.

Many quenchers emit energy through fluorescence reducing the signal tonoise ratio of the probes that contain them and the sensitivity ofassays that utilize them. Such quenchers interfere with the use offluorophores that fluoresce at similar wavelength ranges. This limitsthe number of fluorophores that can be used with such quenchers therebylimiting their usefulness for multiplexed assays which rely on the useof distinct fluorophores in distinct probes that all contain a singlequencher.

Endonucleases (e.g., certain ribonucleases and deoxyribonucleases) areenzymes that cleave the phosphodiester bond within a polynucleotide (DNAor RNA) chain, in contrast to exonucleases, which cleave phosphodiesterbonds at the end of a polynucleotide chain. Typically, a restrictionsite, i.e., a recognition site for an endonuclease, is a palindromicsequence four to six nucleotides long (e.g., TGGATCCA, SEQ ID NO:3).

Endonucleases, found in bacteria and archaea, are thought to haveevolved to provide a defense mechanism against invading viruses. Insidea bacterial host, the restriction enzymes selectively cut up foreign DNAin a process called restriction; host DNA is methylated by amodification enzyme (a methylase) to protect it from the restrictionenzyme's activity. Collectively, these two processes form therestriction modification system. To cut the DNA, a restriction enzymemakes two incisions, once through each sugar-phosphate backbone (i.e.each strand) of the DNA double helix.

Some cells secrete copious quantities of non-specific RNases such as Aand T1. RNases are extremely common, resulting in very short lifespansfor any RNA that is not in a protected environment. Similar torestriction enzymes, which cleave highly specific sequences ofdouble-stranded DNA, a variety of endoribonucleases that recognize andcleave specific sequences of single-stranded RNA have been recentlyclassified.

Present technologies for detection of bacterial pathogens aretime-consuming and expensive because they usually require the isolationand culturing of the bacteria. Also, many of the existing technologiesare toxic and/or use radioactive tracers. Further, technologies forimaging bacterial colonization in humans lack sensitivity. Accordingly,a rapid, inexpensive, non-toxic bacterial-specific assay is needed.

SUMMARY OF THE INVENTION

In certain embodiments, the present invention provides a probe fordetecting bacterial or viral endonucleases (e.g., a ribonuclease or adeoxyribonuclease) comprising an oligonucleotide of 2-30 nucleotides inlength, at least one fluorophore operably linked to the oligonucleotide,and at least one fluorescence quencher operably linked to theoligonucleotide, wherein the oligonucleotide is capable of beingspecifically cleaved by bacterial or viral endonuclease but not by amammalian nuclease or a non-bacterial or non-viral nuclease. In certainembodiments, the oligonucleotide is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30 nucleotides in length.

In certain embodiments, the present invention provides a probe fordetecting bacterial or viral endonuclease comprising an oligonucleotideof 2-30 nucleotides in length, at least one fluorophore operably linkedto the oligonucleotide, and at least one fluorescence quencher operablylinked to the oligonucleotide, wherein the oligonucleotide comprises atleast 4 contiguous nucleotides of CTACGTAG (SEQ ID NO:1) or CUACGUAG(SEQ ID NO:2). In certain embodiments, the oligonucleotide is 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

In certain embodiments, the present invention provides a probe fordetecting bacterial or viral endonuclease comprising an oligonucleotideof 2-30 nucleotides in length, at least one fluorophore operably linkedto the oligonucleotide, and at least one fluorescence quencher operablylinked to the oligonucleotide, wherein the oligonucleotide comprisesCTACGTAG (SEQ ID NO:1) or CUACGUAG (SEQ ID NO:2).

The fluorescence-reporter group and the fluorescence-quencher group areseparated by at least one endonuclease-cleavable residue, e.g., RNA baseor DNA base. Such residues serve as a cleavage domain for endonucleases.In certain embodiments, the oligonucleotide is 10-15 nucleotides inlength. In certain embodiments, the oligonucleotide is 11-13 nucleotidesin length.

In certain embodiments, the oligonucleotide comprises 0-50% purines orany value in between. In certain embodiments the oligonucleotidecomprises 100% pyrimidines. In certain embodiments one or more of thepyrimidines are chemically modified. In certain embodiments, one or moreof the pyrimidines are 2′-O-methyl modified. In certain embodiments, oneor more of the pyrimidines are 2′-fluoro modified. In certainembodiments, one or more of the purines are chemically modified. Incertain embodiments, one or more of the purines are 2′-O-methylmodified. In certain embodiments, one or more of the purines are2′-fluoro modified.

In certain embodiments, the at least one fluorophore is selected fromthe group consisting of the fluorophores listed in Table 1, such as forexample, a fluorophore that has an emission in the near infra-red range.In certain embodiments, the fluorophore is a FAM fluorophore.

In certain embodiments, the at least one fluorescence quencher isselected from the group consisting of the quenchers listed in Table 2.In certain embodiments, the at least one fluorescence quencher is ZENfluorescence quencher and/or Iowa Black fluorescence quencher. Incertain embodiments, the fluorophore is a FAM fluorophores, and the atleast one fluorescence quencher is ZEN and 3IAbRQSp.

In certain embodiments, the probe comprises two oligonucleotides thatare completely self-complementary yielding a double-stranded nucleicacid. In certain embodiments, the oligonucleotide comprises both RNA andDNA. In certain embodiments, the oligonucleotide consists of DNA.

In certain embodiments, the probe consists of56-FAM/fCfUfAfCfGfUfAfG/ZEN/3IAbRQSp (SEQ ID NO: 4).

In certain embodiments, the probe consists ofFAM/TTTTTTTTTTT/ZEN/IAbRQSp/(SEQ ID NO:5), wherein 6-FAM is afluorescein amidite fluorophore, ZEN is a ZEN dark quencher, and IAbRQSpis a Iowa Black dark quencher.

In certain embodiments, the microbial endonuclease is a bacterialendonuclease.

In certain embodiments, the bacterial endonuclease is a Staphylococcusaureus, Staphylococcus epidermidis, Staphylococcus lugdunensis,Staphylococcus saprophyticus, Streptococcus pyogenes, Streptococcusagalactiae, Streptococcus pneumoniae, Streptococcus mutans, Listeriamonocytogenes, Corynebacterium diphtheriae, Bordetella pertussis,Clostridium difficile, Clostridium perfringens, Clostridium botulinum,Enterobacter cloacae, Citrobacter freundii, Borrelia burgdorferi,Treponema pallidum, Bacillus anthracis, Bacillus cereus, Enterococcusfaecalis, Enterococcus faecium, Pseudomonas aeruginosa, Acinetobacterbaumannii, Yersinia pestis, Yersinia pseudotuberculosis, Yersiniaenterocolitica, Klebsiella pneumoniae, Vibrio cholerae, Salmonellaenterica, Salmonella typhi, Escherichia coli, Neisseria gonorrhoeae,Neisseria meningitidis, Mycobacterium tuberculosis, Haemophilusinfluenzae, Legionella pneumophila, Francisella tularensis, Bacteroidesfragilis, Brucella abortus, Mycoplasma fermentans, Mycoplasma pneumonia,Mycoplasma genitalium, and/or Chlamydia trachomatis endonuclease.

In certain embodiments, the bacterial endonuclease is an E. coliendonuclease.

In certain embodiments, the microbial endonuclease is a viralendonuclease.

In certain embodiments, the viral endonuclease is a Cytomegalovirus,Human Herpes Virus 1, 2, 3, 4, 5, 6A, 6B, 7 and/or 8 endonuclease.

In certain embodiments, the present invention provides a method ofdetecting the presence of bacteria or viruses (microbes) in a samplecomprising measuring fluorescence of a sample that has been contactedwith a probe described above, wherein a fluorescence level that isgreater than the fluorescence level of a microbe-free control indicatesthat the sample contains a microbe. In certain embodiments, the testfluorescence level is at least 1-100% greater (or any value in between,e.g., 2%, 10%, 20%, 80% 90%) than the control level.

In certain embodiments, the fluorophore absorbs in the range of 650-850nm.

In certain embodiments, the method is an in vitro assay, and wherein thefluorophore is FAM or Cy5.5.

In certain embodiments, the fluorophore is Cy5, Cy5.5, Cy7, Licor IRDye700, Licor IRDye 800 CW, or Alexa Fluor 647, 660, 680, 750, an/or 790.

In certain embodiments, the fluorophore is FAM, TET, HEX, JOE, MAX, Cy3,or TAMRA and the quencher is IBFQ, BHQ1, BHQ2, or Licor IRDye QC-1.

In certain embodiments, the fluorophore is ROX, Texas Red, Cy5, or Cy5.5and the quencher is IBRQ or BHQ2.

In certain embodiments, the present invention provides a method of invivo detection of a microbial infection in a mammal comprising measuringfluorescence in the mammal, wherein the mammal has been administered aprobe described above, wherein a test fluorescence that is greater thanthe fluorescence level of an uninfected control indicates that thesample has a microbial infection. In certain embodiments, thefluorophore is detectable at a depth of 7-14 cm in the mammal.

In certain embodiments, the present invention provides a method fordetecting bacterial or viral endonuclease activity in a test sample,comprising: (a) contacting the test sample with a probe described above,thereby creating a test reaction mixture, (b) incubating the testreaction mixture for a time sufficient for cleavage of the probe by abacterial or viral endonuclease in the sample; and (c) determiningwhether a detectable fluorescence signal is emitted from the testreaction mixture, wherein emission of a fluorescence signal from thereaction mixture indicates that the sample contains a bacterial or viralendonuclease activity.

In certain embodiments, the present invention provides a method fordetecting a bacterial or viral endonuclease activity in a test sample,comprising: (a) contacting the test sample with a probe described above,thereby creating a test reaction mixture, (b) incubating the testreaction mixture for a time sufficient for cleavage of the substrate bya bacterial or viral endonuclease in the test sample; (c) determiningwhether a detectable fluorescence signal is emitted from the testreaction mixture; (d) contacting a control sample with the substrate,the control sample comprising a predetermined amount of the bacterial orviral endonuclease, thereby creating a control reaction mixture; (e)incubating the control reaction mixture for a time sufficient forcleavage of the substrate by a bacterial or viral endonuclease in thecontrol sample; and (f) determining whether a detectable fluorescencesignal is emitted from the control reaction mixture; wherein detectionof a greater fluorescence signal in the test reaction mixture than inthe control reaction mixture indicates that the test sample containsgreater bacterial or viral endonuclease activity than in the controlsample, and wherein detection of a lesser fluorescence signal in thetest reaction mixture than in the control reaction mixture indicatesthat the test sample contains less bacterial or viral endonucleaseactivity than in the control sample.

In certain embodiments, the predetermined amount of bacterial or viralendonuclease is no bacterial or viral endonuclease, such that detectionof a greater fluorescence signal in the test reaction mixture than inthe control reaction mixture indicates that the test sample containsbacterial or viral endonuclease activity.

In certain embodiments, the method further comprises contacting the testsample with a reaction buffer before or during step (a).

In certain embodiments, the reaction buffer comprises: 50 mM Tris-HCl,pH 8.0, 100 mM NaCl, 12 mM MgCl₂, 1% Triton X-100, 1 mM DTT, and 1×Protease Inhibitor Cocktail.

In certain embodiments, the bacterial or viral endonuclease is E. coliEndonuclease I.

In certain embodiments, the microbe is a bacterium or a virus.

In certain embodiments, the bacterium is Staphylococcus aureus,Staphylococcus epidermidis, Staphylococcus lugdunensis, Staphylococcussaprophyticus, Streptococcus pyogenes, Streptococcus agalactiae,Streptococcus pneumoniae, Streptococcus mutans, Listeria monocytogenes,Corynebacterium diphtheriae, Bordetella pertussis, Clostridiumdifficile, Clostridium perfringens, Clostridium botulinum, Enterobactercloacae, Citrobacter freundii, Borrelia burgdorferi, Treponema pallidum,Bacillus anthracis, Bacillus cereus, Enterococcus faecalis, Enterococcusfaecium, Pseudomonas aeruginosa, Acinetobacter baumannii, Yersiniapestis, Yersinia pseudotuberculosis, Yersinia enterocolitica, Klebsiellapneumoniae, Vibrio cholerae, Salmonella enterica, Salmonella typhi,Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis,Mycobacterium tuberculosis, Haemophilus influenzae, Legionellapneumophila, Francisella tularensis, Bacteroides fragilis, Brucellaabortus, Mycoplasma fermentans, Mycoplasma pneumonia, Mycoplasmagenitalium, and/or Chlamydia trachomatis.

In certain embodiments, the bacterium is E. coli.

In certain embodiments, the microbe is a virus.

In certain embodiments, the virus is a Cytomegalovirus, Human HerpesVirus 1, 2, 3, 4, 5, 6A, 6B, 7 and/or 8 virus.

Accordingly, in certain embodiments, the present invention providesprobe for detecting a microbial endonuclease comprising anoligonucleotide of 2-30 nucleotides in length, a fluorophore operablylinked to the oligonucleotide, and a quencher operably linked to theoligonucleotide, wherein the oligonucleotide comprises one or moremodified pyrimidines, is capable of being cleaved by a microbialnuclease, and has a DNA TT di-nucleotide, DNA AT di-nucleotide, DNA AAdi-nucleotide or DNA TA di-nucleotide positioned at nucleotides 1 and 2,2 and 3 or 3 and 4. In certain embodiments, the present inventionprovides a probe for detecting a microbial endonuclease comprising anoligonucleotide of 2-30 nucleotides in length, a fluorophore operablylinked to the oligonucleotide, and a quencher operably linked to theoligonucleotide, wherein the oligonucleotide comprises one or moremodified pyrimidines, is resistant to cleavage by mammalian nucleases,and has a DNA TT di-nucleotide positioned at nucleotides 1 and 2 or atnucleotides 2 and 3. As defined herein, the term “resistant to cleavageby mammalian nucleases” means that the oligonucleotide is more readilycleaved by a microbial endonuclease than by a mammalian nuclease. Incertain embodiments, the oligonucleotide is cleaved at least 1%, 10%,100%, or greater than 100% more readily by a microbial endonuclease thanby a mammalian nuclease. In certain embodiments, the oligonucleotide is8-15 nucleotides in length and the DNA TT di-nucleotide is positioned atnucleotides 2 and 3. In certain embodiments, the oligonucleotide is 8-11nucleotides in length and the DNA TT di-nucleotide is positioned atnucleotides 2 and 3. In certain embodiments, the oligonucleotide is 4-6nucleotides in length and the DNA TT di-nucleotide is positioned atnucleotides 1 and 2. In certain embodiments, the oligonucleotide is 6nucleotides in length and the DNA TT di-nucleotide is positioned atnucleotides 1 and 2. In certain embodiments, the probe has greaterstability in serum than NMTT probe. In certain embodiments the DNA TTdi-nucleotide consists of unmodified deoxythymidines. In certainembodiments, the nucleotides at positions other than the DNA TTdi-nucleotide are individually selected from A, C, G or U. In certainembodiments, the nucleotides at positions other than the DNA TTdi-nucleotide are modified.

In certain embodiments, the oligonucleotide comprises 0-50% purines orany value in between. In certain embodiments the oligonucleotidecomprises 100% pyrimidines. In certain embodiments one or more of thepyrimidines are chemically modified. In certain embodiments, one or moreof the pyrimidines are 2′-O-methyl modified. In certain embodiments, oneor more of the pyrimidines are 2′-fluoro modified. In certainembodiments, one or more of the purines are chemically modified. Incertain embodiments, one or more of the purines are 2′-O-methylmodified. In certain embodiments, one or more of the purines are2′-fluoro modified. In certain embodiments, the oligonucleotide is RNA.

In certain embodiments, the fluorophore is selected from the groupconsisting of the fluorophores listed in Table 1, such as for example, afluorophore that has an emission in the near infra-red range. In certainembodiments, the quencher is selected from the group consisting of thequenchers listed in Table 2. In certain embodiments, the oligonucleotideis single-stranded.

In certain embodiments, the oligonucleotide comprises both RNA and DNA.

In certain embodiments, the present invention provides anoligonucleotide substrate consisting of

(SEQ ID NO: 7) FAM-T T mU mU mU mU mU mU mU mU mU-ZEN-RQ; (SEQ ID NO: 8)FAM-mU mU T T mU mU mU mU mU mU mU-ZEN-RQ; (SEQ ID NO: 9)FAM-mU mU mU mU T T mU mU mU mU mU-ZEN-RQ; (SEQ ID NO: 10)FAM-mU mU mU mU mU mU mU T T mU mU-ZEN-RQ; (SEQ ID NO: 11)FAM-mU mU mU mU mU mU mU mU mU T T-ZEN-RQ; (SEQ ID NO: 12)FAM-mU mU mU T T mU mU mU-ZEN-RQ; (SEQ ID NO: 13)FAM-mU mU T T mU mU-ZEN-RQ; (SEQ ID NO: 14)FAM-UNA-U UNA-U UNA-U UNA-U T T UNA-U UNA-U UNA-U UNA-U UNA-U-ZEN-RQ;(SEQ ID NO: 15) FAM-mC mC mC mC T T mC mC mC mC mC-ZEN-RQ;(SEQ ID NO: 16) FAM-mU T T mU mU mU mU mU mU mU mU-ZEN-RQ;(SEQ ID NO: 17) FAM-mU mU mU T T mU mU mU mU mU mU-ZEN-RQ;(SEQ ID NO: 18) FAM-mU mG T T mG mU mU mU mU mU mU-ZEN-RQ;(SEQ ID NO: 19) FAM-mU mA T T mA mU mU mU mU mU mU-ZEN-RQ;(SEQ ID NO: 20) FAM-T T mU mU mU mU-ZEN-RQ; (SEQ ID NO: 21)FAM-T T mU mU mU mU mU mU-ZEN-RQ; (SEQ ID NO: 22)FAM-mU T T mU mU mU-ZEN-RQ; (SEQ ID NO: 23)FAM-mU T T mU mU mU mU mU-ZEN-RQ; (SEQ ID NO: 24)FAM-T T T T T T T T T T T-ZEN-RQ; (SEQ ID NO: 25)FAM-T T T T T T-ZEN-RQ; or (SEQ ID NO: 26) FAM-T T T T-ZEN-RQ.

The present invention in certain embodiments further provides a methodof detecting a microbial infection of a sample comprising measuringfluorescence of a sample that has been contacted with a probe describedabove, wherein a fluorescence level that is greater than thefluorescence level of an uninfected control indicates that the samplehas a microbial infection. In certain embodiments, the level is at least1-100% greater than the control level.

In certain embodiments, the method is an in vitro assay. In certainembodiments, the fluorophore is FAM, TET, HEX, JOE, MAX, Cy3, or TAMRAand the quencher is IBFQ, BHQ1 or BHQ2. In certain embodiments, thefluorophore is ROX, Texas Red, Cy5, or Cy5.5 and the quencher is IBRQ orBHQ2.

The present invention in certain embodiments further provides a methodof in vivo detection of a microbial infection in a mammal comprisingmeasuring fluorescence in the mammal, wherein the mammal has beenadministered a probe as described above, wherein a fluorescence levelthat is greater than the fluorescence level of an uninfected controlindicates that the sample has a microbial infection. In certainembodiments, the level is at least 1-100% greater than the controllevel. In certain embodiments, the fluorophore absorbs in the range of650-900 nm. In certain embodiments, the fluorophore is Cy5, Cy5.5, Cy7,Licor IRD700, Cy7.5, Dy780, Dy781, DyLight 800, Licor IRDye 800 CW,Alexa 647, 660, 680, 750, or 790. In certain embodiments, thefluorophore is detectable at a depth of 7-14 cm in the mammal. Incertain embodiments, the microbial infection is a Mycoplasma infection.In certain embodiments, the microbial infection is a Staphylococcusaureus or Streptococcus pneumoniae infection.

In certain embodiments, the present invention provides in vitro assaysfor evaluating the activity of microbial nucleases on various nucleicacid substrates. In certain embodiments the assay evaluates the activityof Mycoplasma nucleases. In certain embodiments the assay evaluates theactivity of Staphylococcus aureus or Streptococcus pneumoniae nucleases.

In certain embodiments, the methods include detection of bacterialcontamination in research laboratories, medical diagnostic applicationsand medical diagnostic imaging.

In certain embodiments, the present invention provides a method fordetecting nuclease (e.g., endonuclease, such as certain ribonucleases ordeoxyribonucleases) activity in a test sample, comprising: (a)selectively inactivating mammalian nucleases in a sample; (b) contactingthe test sample with a substrate, thereby creating a test reactionmixture, wherein the substrate comprises a nucleic acid moleculecomprising: (i) a cleavage domain comprising a single-stranded region ofRNA, the single-stranded region comprising a 2′-fluoro modifiedpyrimidine or 2′-O-methyl modified pyrimidine that renders theoligonucleotide resistant to degradation by mammalian nucleases; (ii) afluorescence reporter group on one side of the internucleotide linkages;and iii. a non-fluorescent fluorescence-quenching group on the otherside of the internucleotide linkages; (c) incubating the test reactionmixture for a time sufficient for cleavage of the substrate by anuclease (e.g., endonuclease, such as certain ribonucleases ordeoxyribonucleases) in the sample; and (d) determining whether adetectable fluorescence signal is emitted from the test reactionmixture, wherein emission of a fluorescence signal from the reactionmixture indicates that the sample contains nuclease (e.g., endonuclease,such as certain ribonucleases or deoxyribonucleases) activity. Asdefined herein, the term “selectively inactivating mammalian nucleases”means that mammalian nucleases present in the sample are reduced atleast 1%, 10%, 100%, as compared to a sample that has not beeninactivated.

In certain embodiments, the present invention provides a method fordetecting nuclease (e.g., endonuclease, such as certain ribonucleases ordeoxyribonucleases) activity in a test sample, comprising: (a)selectively inactivating mammalian nucleases in a sample; (b) contactingthe test sample with a substrate, thereby creating a test reactionmixture, wherein the substrate comprises a nucleic acid moleculecomprising: (i) a cleavage domain comprising a single-stranded region,the single-stranded region of nucleic acid comprising a 2′-fluoromodified pyrimidine or 2′-O-methyl modified pyrimidine that renders theoligonucleotide resistant to degradation by mammalian nucleases; (ii) afluorescence reporter group on one side of the internucleotide linkages;and iii. a non-fluorescent fluorescence-quenching group on the otherside of the internucleotide linkages; (c) incubating the test reactionmixture for a time sufficient for cleavage of the substrate by anuclease activity in the test sample; (d) determining whether adetectable fluorescence signal is emitted from the test reactionmixture; (e) contacting a control sample with the substrate, the controlsample comprising a predetermined amount of nuclease, thereby creating acontrol reaction mixture; (f) incubating the control reaction mixturefor a time sufficient for cleavage of the substrate by a nuclease in thecontrol sample; and (g) determining whether a detectable fluorescencesignal is emitted from the control reaction mixture; wherein detectionof a greater fluorescence signal in the test reaction mixture than inthe control reaction mixture indicates that the test sample containsgreater nuclease activity than in the control sample, and whereindetection of a lesser fluorescence signal in the test reaction mixturethan in the control reaction mixture indicates that the test samplecontains less nuclease activity than in the control sample. In certainembodiments, the nucleic acid is RNA.

In certain embodiments, the present invention provides a method ofdetecting endonuclease (e.g., ribonuclease) activity in a test sample,comprising:

(a) contacting the test sample with a probe or substrate as describedherein to form a digested probe,

(b) collecting the digested probe, and

(c) measuring the fluorescence emitted by the digested probe.

In certain embodiments, the test sample comprises a biological sampleand calcium chloride. In certain embodiments, the biological sample is ablood sample. In certain embodiments, the blood sample is whole blood,serum or plasma. In certain embodiments, the blood sample is notsubjected to a culturing step.

In certain embodiments, the calcium chloride is at a concentration ofabout 5 to 20 mM. In certain embodiments, the calcium chloride is at aconcentration of about 10 mM.

In certain embodiments, the sample has been heated at 55-100° C. for 10seconds to 20 hours to form a heat-treated test sample prior to testing.In certain embodiments, the sample has been heated at about 70 to 95° C.In certain embodiments, the sample has been heated for about 15-30minutes. In certain embodiments, the sample has been heated at about 90°C. for about 20 minutes to form a heat-treated test sample prior totesting.

In certain embodiments, the sample has been clarified after the heatingstep. In certain embodiments, the clarification is by means ofcentrifugation at 1 k to 20 k×g for 10 seconds to 20 minutes after theheating step to form a heat-treated, clarified supernatant test sample.In certain embodiments, the clarification is by means of centrifugationat about 17 k×g for about 10 minutes after the heating step to form aheat-treated, clarified supernatant test sample. In certain embodiments,the clarification is by means of filtration after the heating step toform a heat-treated, clarified supernatant test sample.

In certain embodiments, an endonuclease (e.g, a ribonuclease) present inthe heat-treated test sample has been concentrated prior to testing. Incertain embodiments, the concentration is by means of anaptamer-mediated pull-down. In certain embodiments, the concentration isby means of immunoprecipitation. In certain embodiments, theimmunoprecipitated endonuclease remains bound to an antibody used in theimmunoprecipitation during contact with the probe. In certainembodiments, the immunoprecipitation is by means of anti-micrococcalnuclease antibody-coupled magnetic beads. In certain embodiments, theimmunoprecipitation is specific for a particular microbe. In certainembodiments, the immunoprecipitation is by means of anti-micrococcalnuclease antibody-coupled magnetic beads. In certain embodiments, themagnetic beads are Protein G-coupled magnetic beads.

In certain embodiments, the fluorescence is measured at 485/530 nmexcitation/emission.

In certain embodiments, the endonuclease is a Staphylococcal aureusendonuclease and the probe is NMTT.

In certain embodiments, the endonuclease is a E. coli endonuclease andthe probe is CTACGTAG (SEQ ID NO:1) or CUACGUAG (SEQ ID NO:2).

As used herein, the term “nucleic acid” and “polynucleotide” refers todeoxyribonucleotides or ribonucleotides and polymers thereof in eithersingle- or double-stranded form, composed of monomers (nucleotides)containing a sugar, phosphate and a base that is either a purine orpyrimidine. Unless specifically limited, the term encompasses nucleicacids containing known analogs of natural nucleotides which have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides

“Operably-linked” refers to the association two chemical moieties sothat the function of one is affected by the other, e.g., an arrangementof elements wherein the components so described are configured so as toperform their usual function.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. DNA zymogram identifies Endonuclease 1 as a robust nuclease in arepresentative coliform E. coli strain. Lysates of the indicated E. colistrains were resolved with SDS-PAGE in which salmon sperm DNA wasembedded in the gel matrix. The proteins were allowed to re-nature andthe gel was then incubated at 37° C. to allow digestion. The gel wasstained with SYBR Gold.

FIG. 2. Western blot confirms that Endonuclease 1 protein is absent inEndA KO E. coli strains. Lysates of the indicated E. coli strains wereresolved with SDS-PAGE, transferred to a membrane and probed with arabbit anti-Endonuclease 1 antibody. The box indicates a band of theapproximate molecular weight of Endonuclease 1 that is visible in Cec,Parental, nfi KO and nth KO strains, but not in the EndA KO strains.

FIG. 3. Preference of E. coli nuclease(s) for double-strandedoligonucleotides. The 4×A DNA probe (open bars) or the 4×A DNA probeannealed to a non-fluorescent complementary oligonucleotide (black bars)were incubated with buffer only or with lysates of the indicated strainsof E. coli for 1 hour at 37° C.

FIG. 4. Annealed configuration of self-complementary probes. Oligo 1 isdepicted here (SEQ ID NO: 30). Note the complete self-complementaritythat yields double-stranded nucleic acids. “FAM” indicates the positionof the fluorescein amidite modification. “ZEN” and “RQ” indicate thepositions of the ZEN and Iowa Black RQ fluorescence quenchers.

FIGS. 5A and 5B. Optimal pH and divalent cation for Endonuclease Iactivity. Self-Hybridizing DNA Probe (Oligo 1) was incubated in bufferonly or Coliform E. coli lysates (Cec) for 30 minutes at 37° C. Thebuffer consisted of 50 mM Tris with varying pH, 1% Triton X-100, 1 mMDTT, 1× protease inhibitors, 50 mM NaCl and 1 mM of either CaCl₂ orMgCl₂ as indicated.

FIG. 6. Optimal MgCl₂ concentration for Endonuclease I activity.Self-Hybridizing DNA Probe (Oligo 1) was incubated in buffer only orColiform E. coli lysates (Cec) for 30 minutes at 37° C. The buffersconsisted of 50 mM Tris pH 8, 1% Triton X-100, 1 mM DTT, lx proteaseinhibitors, 50 mM NaCl and the indicated concentrations of MgCl₂.

FIG. 7. Optimal NaCl concentration for Endonuclease I activity.Self-Hybridizing DNA Probe (Oligo 1) was incubated in buffer only orColiform E. coli lysates (Cec) for 30 minutes at 37° C. The buffersconsisted of 50 mM Tris pH 8, 1% Triton X-100, 1 mM DTT, lx proteaseinhibitors, 12 mM MgCl₂ and the indicated concentrations of NaCl. Theoptimized buffer used in subsequent experiments consists of: 50 mM Tris,pH 8, 1% Triton X-100, 1 mM DTT, 1× protease inhibitors, 12 mM MgCl₂,100 mM NaCl.

FIG. 8. Endonuclease I is the nuclease within E. coli lysatesresponsible for Oligo 1 activation. Oligo 1 was incubated with bufferonly (optimized digestion buffer described above) or with lysates of theindicated strains of E. coli for 30 minutes at 37° C. Note that 2distinct strains of E. coli (Keio collection strains 6023 and 8144) inwhich Endonuclease I is deleted were tested.

FIG. 9. Self-hybridizing probes composed of DNA (Oligo 1) and 2′-fluoromodified RNA (Self-Hyb Fl) are activated by nuclease(s) in coliform E.coli (Cec) lysate. Each of the indicated probes was incubated withbuffer only (optimized digestion buffer described above) or withcoliform E. coli lysate for 30 minutes at 37° C.

FIG. 10. Endonuclease I is the nuclease within E. coli lysatesresponsible for Self-Hyb Fl probe activation. The Self-Hyb Fl probe wasincubated with buffer only (optimized digestion buffer described above)or with lysates of the indicated strains of E. coli for 30 minutes at37° C. Note that 2 distinct strains of E. coli (Keio collection strains6023 and 8144) in which Endonuclease I is deleted were tested.

FIG. 11. Coliform E. coli detection sensitivity with nuclease-activatedprobe. Coliform E. coli lysate was diluted and incubated with “Self-HybFl” double-stranded chemically modified RNA probe for 1 hour at 37° C.Each 10 μl reaction was then divided in 3 and fluorescence was measuredwith a plate reader. Numbers on X-axis indicate estimate of bacterialcell number per well of the corresponding data point. The fluorescencelevel of the probe incubated in buffer (i.e., no nucleases) wassubtracted from each value. *The difference between these values and the“buffer only” control is statistically significant.

FIG. 12. Micrococcal nuclease activity is unmasked by heating humanserum that contains the nuclease. Micrococcal nuclease was added tohuman serum to yield the concentrations indicated above. The sampleswere then either unheated, or heated and centrifuged. Unheated samplesand supernatants of heated samples were then incubated with the Poly TTprobe and fluorescence was measured. Note that at concentrations of 2.74nM or less, the activity of the nuclease is substantially less in theunheated serum.

FIG. 13. Increased sensitivity of nuclease activity assay viaimmunoprecipitation of micrococcal nuclease. Micrococcal nuclease wasadded to heparinized human plasma (pooled from healthy donors) to yieldthe concentrations indicated above. The plasma samples were heated,centrifuged and supernatants were divided into two groups. In one set ofsamples, the supernatants were incubated directly with the PolyTT probeprior to plate-reader fluorescence measurements. In the other set ofsamples, micrococcal nuclease was immunoprecipitated ontoantibody-protein G-coupled magnetic beads; the beads were then incubatedwith the PolyTT probe in suspension prior to plate-reader fluorescencemeasurement of the liquid supernatant of this suspension. Note thatconcentrations of micrococcal nuclease as low as 247 attomolar (aM)could be distinguished from the background levels seen with no nucleaseadded (0 M) in the immunoprecipitated samples.

FIG. 14. Detection of micrococcal nuclease in plasma of patients with S.aureus bacteremia. Nuclease activity assays were carried out with plasmaspecimens from S. aureus bacteremic (Infected) and individuals showingno signs of active infections (Presumed Uninfected). Micrococcalnuclease was immunoprecipitated from supernatants of heated andcentrifuged plasma specimens. The immunoprecipitated material wasincubated with the PolyTT quenched fluorescent oligonucleotide probe andfluorescence was measured with a plate-reader. All fluorescence valueswere divided by that of a control sample in which buffer was substitutedfor plasma to yield the Activation Ratios. Data shown are compiled fromseveral independent experiments.

FIGS. 15A-15C. Rapid detection of Mycoplasma-associated nucleaseactivity with chemically modified RNAse substrates. The basis fornuclease detection with RNAse substrates is illustrated in panel A. RNAoligonucleotides (5′-UCUCGUACGUUC-3′ (SEQ ID NO:6) purines in gray andpyrimidines in blue) with chemically modified nucleotides, labeled onthe 5′-ends with FAM are not fluorescent due to the close proximity of a3′-quencher to the FAM. Upon degradation of the oligo, the quencherdiffuses away from the FAM and the FAM exhibits green fluorescence.Mycoplasma-associated nuclease activity is detected with various RNAsesubstrates (panel B). RNAse substrates with the chemically modified RNAcompositions indicated were co-incubated with culture media conditionedby Mycoplasma-free or Mycoplasma contaminated HEK cells for 4 hours at37° C. Fluorescence of these reactions was then measured with afluorescence plate reader. Background fluorescence levels determined bythe fluorescence level of each RNAse substrate incubated in serum-freeunconditioned media have been subtracted from each experimental value.In panel C, the RNAse substrate with 2′-O-methyl-modified pyrimidineswas incubated with the culture supernatant or a lysate prepared frommaterial centrifuged from the supernatants of Mycoplasma-free orMycoplasma-contaminated HEK cells. This assay was carried out asdescribed for B, above, except that the incubation was for only 1 hour.

FIG. 16. 2′-Fluoro pyrimidine and 2′-O-methyl pyrimidine substrates withTriton X-100 lysate of M. fermentans bacteria.

FIG. 17. Degradation activity of Micrococcal Nuclease and EndA Nuclease.Unmodified (RNA and DNA) and modified (2′-Fluoro pyrimidines and2′-O-Methyl pyrimidines) nucleic acid substrates were used to assay thenuclease activity profile of Micrococcal Nuclease (MN) and EndA (H160G)Nuclease. The probes consist of a 12 nucleotide long oligonucleotide,5′-UCUCGUACGUUC-3′ (SEQ ID NO:6), with the chemical modificationsindicated in the figure, flanked by a FAM (5′-modification) and a pairof fluorescence quenchers, “ZEN” and “Iowa Black” (3′-modifications).This approach allows the evaluation of nuclease activity which isindicated by increases in fluorescence upon substrate digestion. 50pmoles of substrate were incubated with MN (1 U/μL) and EndA H160GNuclease (2 μM) in 10 μl total volume. Imadazole was included in theEndA H160G reactions to recapitulate the enzymatic properties of thewildtype enzyme. This mutant version of the enzyme was used because thewt enzyme was toxic to E. coli and could not be produced recombinantlyin large amounts. 50 pmoles of each substrate and buffer were used ascontrols. All reactions were incubated for 30 minutes at 37° C. Afterincubation, 290 μl of buffer supplemented with 10 mM EDTA and 10 mM EGTAwere added to each sample and 95 μl of each sample were loaded intriplicate into a 96-well plate (96F non-treated black microwell plate(NUNC)). Fluorescence intensity was measured with a fluorescencemicroplate reader (Analyst HT; Biosystems).

FIGS. 18A-18D. Digestion of various nucleic acids by bacterialnucleases. Incubation of a 12 nucleotide-long RNA oligo (UCUCGUACGUUC(SEQ ID NO:6) with a 5′-Fam and a 3′-Quencher) with the indicatedmodifications with buffer only, RNAse A (Panel A), MN (1 unit/μl)(Panels A and B) and EndA (20 μM) (Panel B) for 1 hour at 37° C. Panel Cshows digestion of a quenched fluorescent DNA oligo with S. aureusculture supernatants (+ or −MN) incubated for 10 minutes at 37° C.Digestion results in florescence increases in each of the experiments inPanels A-C. Panel D shows the PAGE analysis of a 51 nucleotide-longFAM-labeled (3′-end” RNA oligo with the indicated modifications after 1hour, 37° C. incubation with complete, serum-containing cell culturemedia or with the same media conditioned by HEK cells contaminated withMycoplasma fermentans. Arrow indicates full-length RNA. Modified RNAswere not digested in media conditioned with uncontaminated HEK cells.

FIG. 19. Digestion of oligonucleotide substrates with variousconcentrations of micrococcal nuclease (MN).

FIG. 20. Oligonucleotide substrate plate-reader assays.

FIG. 21. Cultures of the indicated bacteria were grown to stationaryphase. Bacteria were pelleted via centrifugation and nuclease activityof supernatants was measured as described for FIG. 13. To determinebackground levels of probe fluorescence/activation in each of thebacteria-free culture broth preparations used, probes were combined witheach of the indicated broths in addition to PBS and incubated inparallel with the culture supernatant reactions. Incubation time was 15minutes.

FIGS. 22A-22B. Activation of various nucleic acid probes (see Table 4for probe details) by MN, mouse and human serum (A), and S. aureusMN-expressing and MN-negative (Newman and UAMS-1 strains) culturesupernatants (B). 50 picomoles of each of the indicated probes wasincubated with 1 U/μl (positive control) or 0.1 U/μl MN in DPBS(includes physiological levels of calcium and magnesium), or with 90%mouse or human serum (A) or with 90% of culture supernatants of theindicated S. aureus strains (prepared as described in Materials andMethods) for 60 minutes at 37° C. After the incubations, each reactionwas divided into 3 volumes which were read in a fluorescenceplate-reader. Mean fluorescence values of all reactions with a givenprobe were normalized to the mean fluorescence measured with digestionof the probe with 1 U/μl MN. Error bars represent standard deviations ofthe plate-reader values. Background fluorescence subtractions werecarried out (prior to normalization) as follows: The fluorescence ofeach of the probes incubated in DPBS was subtracted from thecorresponding MN-containing reactions. The fluorescence of each of theprobes incubated in DPBS plus the autofluorescence of each serum (mouseor human) was subtracted from the serum-containing reactions. Thefluorescence of each of the probes incubated in unconditioned TSB wassubtracted from the corresponding S. aureus culture supernatantreactions.

FIGS. 23A-F. Activation of the Cy5.5-TT probe by MN in vitro and in micewith MN-expressing S. aureus pyomyositis. For in vitro evaluation of theCy5.5-TT nuclease-activated probe, serial dilutions of the probe werecombined with DPBS or DPBS+1 U/μl MN in 100 μl volumes and incubated at37° C. for 1 hour. Cy5.5 fluorescence was measured for each reaction ina 96-well plate in a Xenogen IVIS 200 imaging system. Controls includeDPBS (left column) and the unquenched TT probe (second column) dilutedin DPBS. To evaluate probe activation in mice with S. aureus-derivedpyomyositis, uninfected mice (n=3 mice) (A), mice with lux+MN-expressingS. aureus (Newman strain) pyomyositis (n=4 mice) (C), and mice withlux+MN-negative S. aureus (Newman strain) pyomyositis (n=4 mice) (D) inthe right thighs were imaged with Cy5.5-channel fluorescence (IVISimaging system) prior to (Bkgd) and after tail vein administration of 3nanomoles of Cy5.5-TT probe. Uninfected mice that received 3 nanomolesof unquenched TT probe (n=3 mice) (B) were imaged in the same manner,but with a shorter exposure time to avoid signal saturation.Luminescence images acquired prior to probe injections (see panels onleft) indicate the location of the infections in C and D. Note probeactivation adjacent to the infection site in C, and minimal probeactivation adjacent to infection site in D. See lookup table signaldisplay ranges (at right of luminescence and right-most fluorescenceimages) for the relationship between pseudocolors and signal strength.Fluorescence display levels are adjusted to show light levels that areabove tissue autofluorescence, fluorescence produced by the unactivatedTT probe or by bleed-through of the luminescence signal into the Cy5.5channel. Time-points listed above fluorescence images indicate the timeelapsed between probe administration and image acquisition. For imagingof probes in mice after sacrifice and dissection, mice with thigh-musclelux+, MN-expressing S. aureus pyomyositis, injected with 3 nanomolesunquenched TT probe (n=4 mice) (E) or TT probe (n=4 mice) (F) weresacrificed 45 minutes after probe injection; organs and skin wereremoved and muscle tissue was imaged with luminescence and the Cy5.5fluorescence channel. Note the lack of overlap between the probefluorescence and bacteria-derived luminescence in E, indicating that theprobe cannot access the infection site. The activated TT probefluorescence is found adjacent to, but not co-localized with, thebacteria-derived luminescence (F). Lookup table signal display ranges ofthe pseudocolored luminescence and fluorescence image data are shown atright.

FIGS. 24A-B. Activation of various nucleic acid probes (see Table 4 forprobe details) by culture supernatants (A) or cell suspensions (B) ofvarious pathogenic bacterial species. 50 picomoles of each of theindicated probes was incubated with 1 U/μl MN (positive control) in DPBSor with 90% of culture supernatants or concentrated and washed cellsuspensions of the indicated bacterial species (prepared as described inExample 6, Materials and Methods) for 60 minutes at 37° C. After theincubations, each reaction was divided into 3 volumes which were read ina fluorescence plate-reader. Mean fluorescence values of all reactionswith a given probe were normalized to the mean fluorescence measuredwith digestion of the probe with 1 U/μl MN. Error bars representstandard deviations of the plate-reader values. Background fluorescencesubtractions were carried out (prior to normalization) as follows: Thefluorescence of each of the probes incubated in DPBS was subtracted fromthe corresponding MN-containing reactions. The fluorescence of each ofthe probes incubated in the appropriate unconditioned culture broth wassubtracted from the corresponding culture supernatant reactions. Thefluorescence of each of the probes incubated in DPBS plus theautofluorescence of each appropriate bacterial suspension was subtractedfrom each bacterial suspension reaction.

FIG. 25. Comparison of the activation kinetics (digestion withmicrococcal nuclease (MN)) of 11 mer quenched fluorescentoligonucleotide probes with a pair of T's in various positions. Note themaximal activation of the probes with the TT closest to the 5′-end ofthe probe (i.e., positions 1&2, 3&4, 5&6) at the earliest time-point.Also, note that the non-T nucleotides consist of 2′-O-methyl modifiedU's in all of the probes except the NMTT probe, in which a variety of2′-O-methyl nucleotides is used. The differences between the NMTT probeand the others could thus be due to these differences in addition to theTT position.

FIG. 26. Comparison of the activation kinetics (digestion withmicrococcal nuclease (MN)) of 3×11 mer quenched fluorescentoligonucleotide probes with a pair of T's in different positions. Notethat these probes were maximally activated at the earliest time-pointmeasured when a higher concentration of the nuclease (0.00625 U/μl) wasused. The 10-fold lower concentration of the enzyme used here provided ameans of identifying the most sensitive probe for MN.

FIG. 27. Comparison of the activation kinetics (digestion withmicrococcal nuclease (MN)) of 11 mer quenched fluorescentoligonucleotide probes with a pair of T's in different positions.Inclusion of 2 additional probes with the TT positioned near the 5′-end(P2&3 TT Probe and P4&5 TT Probe) enabled a more precise determinationof the optimal position for this pair of nucleotides.

FIG. 28. Comparison of the activation kinetics (digestion withmicrococcal nuclease (MN)) of 6 mer and 8 mer quenched fluorescentoligonucleotide probes with a pair of T's in different positions. Noneof these probes was as sensitive as the best 11 mer probe which yieldedsimilar activation kinetics to the best of these probes when 5-fold lessenzyme was used. The optimal position for the TT appears to be 1 &2 forthe 6 mer and 2&3 for the 8 mer. Note also that the 8 mers are moresensitive to MN than the 6 mers.

FIG. 29. Comparison of the activation kinetics (digestion withmicrococcal nuclease (MN)) of 3×8 mer quenched fluorescentoligonucleotide probes with a pair of T's in different positions. Thelower concentration of MN enabled identification of the best of these 3probes, which were difficult to distinguish when higher concentrationswere used. TT positioned in nucleotides 2&3 appears optimal for the 8mer probe length.

FIG. 30. Serum stability and activation by MN in serum of the NMTT probeand 2 probe variants. All 3 probes are 11 mers with TT in positions 5 &6. The additional nucleotide positions are different, but all of theseadditional nucleotides are 2′-O-methyl modified. The ratios betweenfluorescence of probes digested by MN in serum versus that of probesincubated in serum without MN (right panel) are a measure of signal tobackground. These results do not show a substantial difference among theprobes. This data suggests that the additional, non-T nucleotides do nothave an important impact on this measure of probe performance.

FIG. 31. Serum stability and activation by MN in serum of the NMTT probe(included as a control) and 3 probe variants in which the nucleotidesimmediately flanking the TT are variable (the remaining nucleotides inall 3 of these probes are 2′-O-methyl U's). Among the 3 probes withvariable flanking nucleotides, the ratios of fluorescence of MN-digestedversus serum only incubation ranged from ˜7 to ˜14. These flankingnucleotides could therefore have an impact on this measure of probeperformance.

FIG. 32. Serum stability and activation by MN in serum of the NMTT probe(included as a control) and 3 probes of variable length composed of astring of T's. Note the ˜70-fold ratio of the MN-digested (in serum)versus serum-only fluorescence of the Poly TT 4 mer (right panel).Despite the fact that the oligonucleotide portion of this probe consistsof unmodified nucleotides (DNA T's), it is very stable in serum (leftpanel).

FIG. 33. Comparison of the activation kinetics (digestion withmicrococcal nuclease (MN)) of 3 quenched fluorescent oligonucleotideprobes of variable length composed of a string of T's. Note: the shorterthe probe, the less sensitive it is to MN.

FIG. 34. Serum stability and activation by MN in serum of the NMTT probe(included as a control) and 2 probes of variable length composed of TTflanked by several 2′-O-methyl U's. Note the greater than ˜50-fold ratioof the MN-digested (in serum) versus serum-only fluorescence of the TTProbe 6 mer (right panel). While this ratio is superior to that of theNMTT probe, the TT Probe 6 mer, like the other short probes, was foundto be less sensitive than the NMTT probe to micrococcal nucleasedigestion in kinetics assays.

FIG. 35. Serum stability and activation by MN in serum of the NMTT probe(included as a control) and 2 probes with a TT flanked by several2′-O-methyl C's (P5&6 TT mC Probe) or by several unlocked nucleic acidU's (UNA P5&6 TT Probe). Both of these alternative probe configurationsis digested by MN in serum. However, neither performs as well as theNMTT probe in this assay.

FIG. 36. Digestion of TT probes (oligonucleotide portion is identical tothe NMTT probe) in which the indicated NIR fluorophores are used inplace of FAM and the QC-1 quencher is used at the 3′-end. Probes wereincubated in buffer only or buffer plus nuclease and ratio of digestedversus buffer only fluorescence is plotted. Digests were carried outwith or without heparin to examine the effect of this compound prior tomeasurement of probe digestion in heparinized blood. Note that allratios indicate some degree of probe activation via digestion. (3-digitnumbers following some of the probe names indicate synthesis variants ofthe probes. Upon purification of the probes with HPLC, differentpurification peaks were separately collected and assigned thesenumbers.)

FIG. 37. Activation of near-infrared probes in whole blood. Ratios offluorescence of probe incubated in whole mouse blood plus micrococcalnuclease for 60 minutes divided by fluorescence of probe incubated inwhole mouse blood without nuclease for 1 minute. Higher ratios indicatebetter probe performance.

FIG. 38. Fluorescence Ratios of TT Probes (with NIR fluorophore/quencherpairs) incubated in blood for 60 minutes versus 1 minute. As no nucleasewas added, this is just a measure of probe stability in blood. The lowerthe ratio, the greater the stability.

DETAILED DESCRIPTION OF THE INVENTION

Close to 1 billion people currently depend on contaminated sources ofwater, a major underlying cause of diarrheal diseases which account forapproximately 4% of disease burden globally (Connelly, J. T. & Baeumner,A. J. Biosensors for the detection of waterborne pathogens. Anal BioanalChem 402, 117-127 (2012)). Pathogenic microbial contaminants of drinkingwater include viruses, bacteria and parasites. The most common source ofbacterial contamination of water supplies is animal and/or human feces.Current testing for fecal contamination depends on detection of“indicator” organisms, such as coliform Escherichia coli (E. coli),which are used because they are present in feces in great abundance andare thus easier to detect than many pathogens. Methods used fordetecting coliform E. coli have a variety of limitations, including: 1)sensitivity for only a subset of E. coli strains, 2) time-intensivenature of the methods, 3) need for transport of water samples toappropriately equipped laboratories (Connelly, J. T. & Baeumner, A. J.Biosensors for the detection of waterborne pathogens. Anal Bioanal Chem402, 117-127 (2012)). For instance, the Colilert® and Colisure® coliformE. coli detecting kits (of IDEXX Laboratories), which use traditionalenzyme-detection methods for microbial detection, require 24-48 hours ofculture and do not detect important pathogenic forms of E. coli,including the O157:H7 strain (Straub, T. M. & Chandler, D. P. Towards aunified system for detecting waterborne pathogens. J Microbiol Methods53, 185-197 (2003)). PCR-based methods are capable of preciseidentification of bacterial species and strains present, but thesemethods are also time-consuming and require laboratories withappropriate technical infrastructure. Considering the limitations ofexisting technologies, a novel method that rapidly and specificallydetects coliform E. coli in water samples in the field could be adisruptive technology in this market.

In certain embodiments, the present invention provides shortoligonucleotide probes (Substrates) composed of chemically modified DNAor RNA flanked with at least one fluorophore on one end and at least onefluorescence quencher on the other end. Upon cleavage of the probes bynucleases (e.g., endonuclease), the fluorophore diffuses away from thequencher and exhibits fluorescence. These probes are not cleaved bymammalian nucleases, but are cleaved by nucleases produced by variousbacteria, including pathogenic bacteria such as Escherichia coli (E.coli). The probes can thus be used to detect the presence of E. coli inbiological samples such as blood serum, cell cultures, and food, and invivo, and in environmental samples, such as water.

In certain embodiments, the present invention provides shortoligonucleotide probes (Substrates) composed of chemically modified RNAflanked with a fluorophore on one end and a fluorescence quencher on theother end. Upon cleavage of the probes by nucleases (e.g.,endonucleases, such as certain ribonucleases), the fluorophore diffusesaway from the quencher and exhibits fluorescence. These probes are notcleaved by mammalian nucleases, but are cleaved by nucleases produced byvarious bacteria, including pathogenic bacteria such as Staphylococcusaureus, Streptococcus pneumoniae or Mycoplasma. The probes can thus beused to detect the presence of bacteria in biological samples such asblood serum, cell cultures, and food, and in vivo.

The present invention relates to methods for detecting nuclease (e.g.,endonuclease) activity in a sample, comprising: 1) optionally,selectively inactivating mammalian nucleases in a sample and incubatinga synthetic Substrate or mixture of Substrates in the sample, for a timesufficient for cleavage of the Substrates(s) by a nuclease enzyme,wherein the Substrate(s) comprises a single-stranded nucleic acidmolecule containing at least one ribonucleotide or deoxyribonucleotideresidue at an internal position that functions as a nuclease (e.g.,endonuclease) cleavage site (and in certain embodiments a 2′-fluoromodified pyrimidine or 2′-O-methyl modified pyrimidine that renders theoligonucleotide resistant to degradation by mammalian nucleases), afluorescence reporter group on one side of the cleavage sites, and afluorescence-quenching group on the other side of the cleavage site, and2) visual detection of a fluorescence signal, wherein detection of afluorescence signal indicates that a nuclease (e.g., endonuclease)cleavage event has occurred, and, therefore, the sample containsnuclease (e.g., endonuclease) activity. The compositions of theinvention are also compatible with other detection modalities (e.g.,fluorometry).

The Substrate oligonucleotide of the invention comprises a fluorescentreporter group and a quencher group in such physical proximity that thefluorescence signal from the reporter group is suppressed by thequencher group. Cleavage of the Substrate with a nuclease (e.g.,endonuclease) enzyme leads to strand cleavage and physical separation ofthe reporter group from the quencher group. Separation of reporter andquencher eliminates quenching, resulting in an increase in fluorescenceemission from the reporter group. When the quencher is a so-called “darkquencher”, the resulting fluorescence signal can be detected by directvisual inspection (provided the emitted light includes visiblewavelengths). Cleavage of the Substrate compositions described in thepresent invention can also be detected by fluorometry.

In one embodiment, the synthetic Substrate is an oligonucleotidecomprising ribonucleotide residues. The synthetic Substrate can also bea chimeric oligonucleotide comprising RNase-cleavable, e.g., RNA,residues, or modified RNase-resistant RNA residues. In certainembodiments, Substrate composition is such that cleavage is aribonuclease-specific event and that cleavage by enzymes that arestrictly deoxyribonucleases does not occur.

In one embodiment, the synthetic Substrate is a chimeric oligonucleotidecomprising ribonucleotide residue(s) and modified ribonucleotideresidue(s). In one embodiment, the synthetic Substrate is a chimericoligonucleotide comprising ribonucleotide residues and 2′-O-methylribonucleotide residues. In one embodiment, the synthetic Substrate is achimeric oligonucleotide comprising 2′-O-methyl ribonucleotide residuesand one or more of each of the four ribonucleotide residues, adenosine,cytosine, guanosine, and uridine. Inclusion of the four distinctribonucleotide bases in a single Substrate allows for detection of anincreased spectrum of endonuclease enzyme activities by a singleSubstrate oligonucleotide.

In one embodiment, the synthetic Substrate is an oligonucleotidecomprising deoxyribonucleotide residues. The synthetic Substrate canalso be a chimeric oligonucleotide comprising DNase-cleavable, e.g.,DNA, residues, or modified RNase-resistant RNA residues. Substratecomposition is such that cleavage is a deoxyribonuclease-specific eventand that cleavage by enzymes that are strictly ribonucleases does notoccur.

In one embodiment, the synthetic Substrate is a chimeric oligonucleotidecomprising deoxyribonucleotide residue(s) and modified ribonucleotideresidue(s). In one embodiment, the synthetic Substrate is a chimericoligonucleotide comprising deoxyribonucleotide residues and 2′-O-methylribonucleotide residues. In one embodiment, the synthetic Substrate is achimeric oligonucleotide comprising 2′-O-methyl ribonucleotide residuesand one or more of each of the four deoxyribonucleotide residues,deoxyadenosine, deoxycytosine, deoxyguanosine, and deoxythymidine.Inclusion of the four distinct deoxyribonucleotide bases in a singleSubstrate allows for detection of an increased spectrum ofdeoxyribonuclease enzyme activities by a single Substrateoligonucleotide.

To enable visual detection methods, the quenching group is itself notcapable of fluorescence emission, being a “dark quencher”. Use of a“dark quencher” eliminates the background fluorescence of the intactSubstrate that would otherwise occur as a result of energy transfer fromthe reporter fluorophore. In one embodiment, the fluorescence quenchercomprises dabcyl (4-(4′-dimethylaminophenylazo)benzoic acid). In oneembodiment, the fluorescence quencher is comprised of QSY™-7 carboxylicacid, succinimidyl ester(N,N′-dimethyl-N,N′-diphenyl-4-((5-t-butoxycarbonylaminopentyl)aminocarbonyl) piperidinylsulfonerhodamine; a diarylrhodamine derivative fromMolecular Probes, Eugene, Oreg.). Any suitable fluorophore may be usedas reporter provided its spectral properties are favorable for use withthe chosen quencher. A variety of fluorophores can be used as reporters,including but not limited to, fluorescein, tetrachlorofluorescein,hexachlorofluorescein, rhodamine, tetramethylrhodamine, Cy-dyes, TexasRed, Bodipy dyes, and Alexa dyes.

In certain embodiments, the method of the invention proceeds in multiplesteps. In certain embodiments, mammalian nucleases are selectivelyinactivated in a sample. Next, the test sample is mixed with theSubstrate reagent and incubated. Substrate can be mixed alone with thetest sample or will be mixed with an appropriate buffer, e.g., one of acomposition as described herein. Next, visual detection of fluorescenceis performed. As fluorescence above background indicates fluorescenceemission of the reaction product, i.e. the cleaved Substrate, detectionof such fluorescence indicates that RNase activity is present in thetest sample. The method provides that this step can be done withunassisted visual inspection. In particular, visual detection can beperformed using a standard ultraviolet (UV) light source of the kindfound in most molecular biology laboratories to provide fluorescenceexcitation. Substrates of the invention can also be utilized in assayformats in which detection of Substrate cleavage is done using amulti-well fluorescence plate reader or a tube fluorometer.

The present invention further features kits for detecting nuclease(e.g., endonuclease) activity comprising a Substrate nucleic acid(s) andinstructions for use. Such kits may optionally contain one or more of: apositive control nuclease (e.g., endonuclease), RNase-free water, and abuffer. It is also provided that the kits may include RNase-freelaboratory plasticware, for example, thin-walled, UV transparentmicrotubes for use with the visual detection method and/or multiwellplates for use with plate-fluorometer detection methods in ahigh-throughput format.

Accordingly, the present invention provides a method for detectingnuclease (e.g., endonuclease) activity in a test sample (optionally, inwhich mammalian nuclease will have been selectively inactivated in thesample), comprising: (a) contacting the test sample with a substrate,thereby creating a test reaction mixture, wherein the substratecomprises a nucleic acid molecule comprising (i) a cleavage domaincomprising a single-stranded region, the single-stranded regioncomprising at least one internucleotide linkage (and in certainembodiments a 2′-fluoro modified pyrimidine or 2′-O-methyl modifiedpyrimidine that renders the oligonucleotide resistant to degradation bymammalian nucleases); (ii) a fluorescence reporter group on one side ofthe internucleotide linkage; and (iii) a non-fluorescentfluorescence-quenching group on the other side of the internucleotidelinkage; (b) incubating the test reaction mixture for a time sufficientfor cleavage of the substrate by a endonuclease in the sample; and (c)determining whether a detectable fluorescence signal is emitted from thetest reaction mixture, wherein emission of a fluorescence signal fromthe reaction mixture indicates that the sample contains endonucleaseactivity.

While the methods of the invention can be practiced without the use of acontrol sample, in certain embodiments of the invention it is desirableto assay in parallel with the test sample a control sample comprising aknown amount of RNase activity. Where the control sample is used as anegative control, the control sample, in some embodiments, contains nodetectable RNase activity. Thus, the present invention further providesa method for detecting endonuclease activity in a test sample,comprising: (a) contacting the test sample with a substrate, therebycreating a test reaction mixture, wherein the substrate comprises anucleic acid molecule comprising: (i) a cleavage domain comprising asingle-stranded region, the single-stranded region comprising at leastone internucleotide linkage (and in certain embodiments a 2′-fluoromodified pyrimidine or 2′-O-methyl modified pyrimidine that renders theoligonucleotide resistant to degradation by mammalian nucleases); (ii) afluorescence reporter group on one side of the internucleotide linkage;and (iii) a non-fluorescent fluorescence-quenching group on the otherside of the internucleotide linkage; (b) incubating the test reactionmixture for a time sufficient for cleavage of the substrate by anuclease (e.g., endonuclease) activity in the test sample; (c)determining whether a detectable fluorescence signal is emitted from thetest reaction mixture; (d) contacting a control sample with thesubstrate, the control sample comprising a predetermined amount ofnuclease (e.g., endonuclease), thereby creating a control reactionmixture; (e) incubating the control reaction mixture for a timesufficient for cleavage of the substrate by a nuclease (e.g.,endonuclease) in the control sample; (f) determining whether adetectable fluorescence signal is emitted from the control reactionmixture; wherein detection of a greater fluorescence signal in the testreaction mixture than in the control reaction mixture indicates that thetest sample contains greater nuclease (e.g., endonuclease) activity thanin the control sample, and wherein detection of a lesser fluorescencesignal in the test reaction mixture than in the control reaction mixtureindicates that the test sample contains less nuclease (e.g.,endonuclease) activity than in the control sample. In one embodiment,the predetermined amount of nuclease (e.g., endonuclease) is nonuclease, such that detection of a greater fluorescence signal in thetest reaction mixture than in the control reaction mixture indicatesthat the test sample contains nuclease (e.g., endonuclease) activity.

The methods of the invention can further entail contacting the testsample with a buffer before or during step (a).

The present invention further provides compositions and kits forpracticing the present methods. Thus, in certain embodiments, thepresent invention provides a nucleic acid comprising: (a) a cleavagedomain comprising a single-stranded region, the single-stranded regioncomprising at least one internucleotide linkage (and in certainembodiments a 2′-fluoro modified pyrimidine or 2′-O-methyl modifiedpyrimidine that renders the oligonucleotide resistant to degradation bymammalian nucleases); (b) a fluorescence reporter group on one side ofthe internucleotide linkage; and (c) a non-fluorescentfluorescence-quenching group on the other side of the internucleotidelinkage. In other embodiments, the present invention provides a kitcomprising: (a) in one container, a substrate, the substrate comprisinga nucleic acid molecule comprising a single stranded region, thesingle-stranded region comprising: (i) a cleavage domain comprising asingle-stranded region, the single-stranded region comprising at leastone internucleotide linkage 3′ to an adenosine residue, at least oneinternucleotide linkage 3′ to a cytosine residue, at least oneinternucleotide linkage 3′ to a guanosine residue, and at least oneinternucleotide linkage 3′ to a uridine residue, and wherein thecleavage domain does not comprise a deoxyribonuclease-cleavableinternucleotide linkage; (ii) a fluorescence reporter group on one sideof the internucleotide linkages; and (iii) a non-fluorescentfluorescence-quenching group on the other side of the internucleotidelinkages.

In one embodiment of the foregoing methods and compositions, the singlestranded region of the cleavage domain comprises at least oneinternucleotide linkage 3′ to an adenosine residue, at least oneinternucleotide linkage 3′ to a cytosine residue, at least oneinternucleotide linkage 3′ to a guanosine residue, and at least oneinternucleotide linkage 3′ to a uridine residue. In one embodiment, thecleavage domain does not comprise a deoxyribonuclease-cleavableinternucleotide linkage. In yet another referred embodiment, the singlestranded region of the cleavage domain comprises at least oninternucleotide linkage 3′ to an adenosine residue, at least oneinternucleotide linkage 3′ to a cytosine residue, at least oneinternucleotide linkage 3′ to a guanosine residue, and at least oneinternucleotide linkage 3′ to a uridine residue and the cleavage domaindoes not comprise a deoxyribonuclease-cleavable internucleotide linkage.

In one embodiment of the foregoing methods and compositions, the singlestranded region of the cleavage domain comprises at least oneinternucleotide linkage 3′ to a deoxyadenosine residue, at least oneinternucleotide linkage 3′ to a deoxycytosine residue, at least oneinternucleotide linkage 3′ to a deoxyguanosine residue, and at least oneintemucleotide linkage 3′ to a deoxythymidine residue. In oneembodiment, the cleavage domain does not comprise aribonuclease-cleavable internucleotide linkage. In yet another referredembodiment, the single stranded region of the cleavage domain comprisesat least one internucleotide linkage 3″ to a deoxyadenosine residue, atleast one internucleotide linkage 3′ to a deoxycytosine residue, atleast one intemucleotide linkage 3′ to a deoxyguanosine residue, and atleast one internucleotide linkage 3′ to a deoxythymidine residue and thecleavage domain does not comprise a ribonuclease-cleavableinternucleotide linkage.

With respect to the fluorescence quenching group, any compound that is adark quencher can be used in the methods and compositions of theinvention. Numerous compounds are capable of fluorescence quenching,many of which are not themselves fluorescent (i.e., are dark quenchers.)In one embodiment, the fluorescence-quenching group is anitrogen-substituted xanthene compound, a substituted4-(phenyldiazenyl)phenylamine compound, or a substituted4-(phenyldiazenyl)naphthylamine compound. In certain specific modes ofthe embodiment, the fluorescence-quenching group is4-(4′-dimethylaminophenylazo)benzoic acid),N,N′-dimethyl-N,N′-diphenyl-4-((5-t-butoxycarbonylaminopentyl)aminocarbonyl) piperidinylsulfonerhodamine (sold as QSY-7™ by MolecularProbes, Eugene, Oreg.), 4′,5′-dinitrofluorescein, pipecolic acid amide(sold as QSY-33™ by Molecular Probes, Eugene, Oreg.)4-[4-nitrophenyldiazinyl]phenylamine, or4-[4-nitrophenyldiazinyl]naphthylamine (sold by Epoch Biosciences,Bothell, Wash.). In other specific modes of the embodiment, thefluorescence-quenching group is Black-Hole Quenchers™ 1, 2, or 3(Biosearch Technologies, Inc.).

In certain embodiments, the fluorescence reporter group is fluorescein,tetrachlorofluorescein, hexachlorofluorescein, rhodamine,tetramethylrhodamine, a Cy dye, Texas Red, a Bodipy dye, or an Alexadye.

With respect to the foregoing methods and compositions, the fluorescencereporter group or the fluorescence quenching group can be, but is notnecessarily, attached to the 5′-terminal nucleotide of the substrate.

The nucleic acids of the invention, including those for use assubstrates in the methods of the invention, in certain embodiments aresingle-stranded RNA molecule. In other embodiments, the nucleic acids ofthe invention are chimeric oligonucleotides comprising a nucleaseresistant modified ribonucleotide residue. Exemplary RNase resistantmodified ribonucleotide residues include 2′-O-methyl ribonucleotides,2′-methoxyethoxy ribonucleotides, 2′-O-allyl ribonucleotides,2′-O-pentyl ribonucleotides, and 2′-O-butyl ribonucleotides. In one modeof the embodiment, the modified ribonucleotide residue is at the5′-terminus or the 3′-terminus of the cleavage domain. In yet otherembodiments, the nucleic acids of the invention are chimericoligonucleotides comprising a deoxyribonuclease resistant modifieddeoxyribonucleotide residue. In specific modes of the embodiments, thedeoxyribonuclease resistant modified deoxyribonucleotide residue is aphosphotriester deoxyribonucleotide, a methylphosphonatedeoxyribonucleotide, a phosphoramidate deoxyribonucleotide, aphosphorothioate deoxyribonucleotide, a phosphorodithioatedeoxyribonucleotide, or a boranophosphate deoxyribonucleotide. In yetother embodiments of the invention, the nucleic acids of the inventioncomprise a ribonuclease-cleavable modified ribonucleotide residue.

The nucleic acids of the invention, including those for use assubstrates in the methods of the invention, are at least 3 nucleotidesin length, such as 5-30 nucleotides in length. In certain specificembodiments, the nucleic acids of the invention are 5-20, 5-15, 5-10,7-20, 7-15 or 7-10 nucleotides in length.

In certain embodiments, the fluorescence-quenching group of the nucleicacids of the invention is 5′ to the cleavage domain and the fluorescencereporter group is 3′ to the cleavage domain. In a specific embodiment,the fluorescence-quenching group is at the 5′ terminus of the substrate.In another specific embodiment, the fluorescence reporter group is atthe 3′ terminus of the substrate.

In certain embodiments, the fluorescence reporter group of the nucleicacids of the invention is 5′ to the cleavage domain and thefluorescence-quenching group is 3′ to the cleavage domain. In a specificembodiment, the fluorescence reporter group is at the 5′ terminus of thesubstrate. In another specific embodiment, the fluorescence-quenchinggroup is at the 3′ terminus of the substrate.

In one embodiment of the invention, a nucleic acid of the inventioncomprising the formula: 5′-N₁-n-N₂-3′, wherein: (a) “N₁” represents zeroto five 2′-modified ribonucleotide residues; (b) “N₂” represents one tofive 2′-modified ribonucleotide residues; and (c) “n” represents one toten, such as four to ten unmodified ribonucleotide residues. In acertain specific embodiment, “N₁” represents one to five 2′-modifiedribonucleotide residues. In certain modes of the embodiment, thefluorescence-quenching group or the fluorescent reporter group isattached to the 5′-terminal 2′-modified ribonucleotide residue of N₁.

In the nucleic acids of the invention, including nucleic acids with theformula: 5′-N₁-n-N₂-3′, the fluorescence-quenching group can be 5′ tothe cleavage domain and the fluorescence reporter group is 3′ to thecleavage domain; alternatively, the fluorescence reporter group is 5′ tothe cleavage domain and the fluorescence-quenching group is 3′ to thecleavage domain.

With respect to the kits of the invention, in addition to comprising anucleic acid of the invention, the kits can further comprise one or moreof the following: an endonuclease (e.g, a ribonuclease);endonuclease-free water (e.g., ribonuclease-free water), a buffer, andendonuclease-free laboratory plasticware (e.g., ribonuclease-freelaboratory plasticware).

“Probe” or “Substrate” Oligonucleotides

Compositions of the invention comprise synthetic oligonucleotideSubstrates that are substrates for nuclease (e.g., endonuclease)enzymes. Substrate oligonucleotides of the invention comprise: 1) one ormore nuclease-cleavable bases, e.g., RNA bases, some or all of whichfunction as scissile linkages, 2) a fluorescence-reporter group and afluorescence-quencher group (in a combination and proximity that permitsvisual FRET-based fluorescence quenching detection methods), and 3) mayoptionally contain RNase-resistant modified RNA bases,nuclease-resistant DNA bases, or unmodified DNA bases. Syntheticoligonucleotide RNA-DNA chimeras wherein the internal RNA bonds functionas a scissile linkage are described in U.S. Pat. Nos. 6,773,885 and7,803,536. The fluorescence-reporter group and the fluorescence-quenchergroup are separated by at least one RNAse-cleavable residue, e.g., RNAbase. Such residues serve as a cleavage domain for endonucleases (e.g,ribonucleases).

In certain embodiments, the substrate oligonucleotide probes aresingle-stranded or double-stranded oligoribonucleotides. In certainembodiments, the oligonucleotide probes are composed of modifiedoligoribonucleotides. The term “modified” encompasses nucleotides with acovalently modified base and/or sugar. For example, modified nucleotidesinclude nucleotides having sugars which are covalently attached to lowmolecular weight organic groups other than a hydroxyl group at the 3′position and other than a phosphate group at the 5′ position. Thusmodified nucleotides may also include 2′ substituted sugars such as2′-O-methyl-; 2-O-alkyl; 2-O-allyl; 2′-S-alkyl; 2′-S-allyl; 2′-fluoro-;2′-halo or 2-azido-ribose, carbocyclic sugar analogues a-anomericsugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranosesugars, furanose sugars, and sedoheptulose. In certain embodiments, theSubstrate includes, but is not limited to, 2′-O-methyl RNA,2′-methoxyethoxy RNA, 2′-O-allyl RNA, 2′-O-pentyl RNA, and 2′-O-butylRNA. In certain embodiments, the substrate is an RNA-2′-O-methyl RNAoligonucleotide having the general structure 5′ r-NnN-q 3′, where ‘N’represents from about one to five 2′-modified ribonucleotide residues,‘n’ represents one to ten unmodified ribonucleotide residues, ‘r’represents a fluorescence reporter group, and ‘q’ represents afluorescence quencher group. The 5′- and 3′-position of reporter andquencher are interchangeable. In one embodiment, the fluorescencereporter group and the fluorescence quencher group are positioned at ornear opposing ends of the molecule. It is not important which group isplaced at or near the 5′-end versus the 3′-end. It is not required thatthe reporter and quencher groups be end modifications, howeverpositioning these groups at termini simplifies manufacture of theSubstrate. The fluorescence reporter group and the fluorescence quenchergroup may also be positioned internally so long as an RNA scissilelinkage lies between reporter and quencher.

Modified nucleotides are known in the art and include, by example andnot by way of limitation, alkylated purines and/or pyrimidines; acylatedpurines and/or pyrimidines; or other heterocycles. These classes ofpyrimidines and purines are known in the art and include,pseudoisocytosine; N4, N4-ethanocytosine; 8-hydroxy-N6-methyladenine;4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil; 5-fluorouracil;5-bromouracil; 5-carboxymethylaminomethyl-2-thiouracil;5-carboxymethylaminomethyl uracil; dihydrouracil; inosine;N6-isopentyl-adenine; 1-methyladenine; 1-methylpseudouracil;1-methylguanine; 2,2-dimethylguanine; 2-methyladenine; 2-methylguanine;3-methylcytosine; 5-methylcytosine; N6-methyladenine; 7-methylguanine;5-methylaminomethyl uracil; 5-methoxy amino methyl-2-thiouracil;β-D-mannosylqueosine; 5-methoxycarbonylmethyluracil; 5-methoxyuracil;2-methylthio-N6-isopentenyladenine; uracil-5-oxyacetic acid methylester; psueouracil; 2-thiocytosine; 5-methyl-2 thiouracil, 2-thiouracil;4-thiouracil; 5-methyluracil; N-uracil-5-oxyacetic acid methylester;uracil 5-oxyacetic acid; queosine; 2-thiocytosine; 5-propyluracil;5-propylcytosine; 5-ethyluracil; 5-ethylcytosine; 5-butyluracil;5-pentyluracil; 5-pentylcytosine; and 2,6,-diaminopurine;methylpsuedouracil; 1-methylguanine; 1-methylcytosine.

The oligonucleotides of the invention are synthesized using conventionalphosphodiester linked nucleotides and synthesized using standard solidor solution phase synthesis techniques which are known in the art.Linkages between nucleotides may use alternative linking molecules. Forexample, linking groups of the formula P(O)S, (thioate); P(S)S,(dithioate); P(O)NR′2; P(O)R; P(O)OR6; CO; or CONR′2 wherein R is H (ora salt) or alkyl (1-12C) and R6 is alkyl (1-9C) is joined to adjacentnucleotides through —O— or —S—.

In certain embodiments of the present invention, the oligonucleotideshave additional modifications, such as 2′O-methyl modification of thepyrimidines. In other embodiments, all of the nucleotides in theoligonucleotides are 2′O-methyl modified. Alternatively, thepyrimidines, or all the nucleotides, may be modified with 2′-fluoros(both pyrimidines and purines).

The oligonucleotides are short, such as between 2-30 nucleotides inlength (or any value in between). In certain embodiments, thatoligonucleotide is between 8-15 nucleotides in length. In certainembodiments, that oligonucleotide is between 11-13 nucleotides inlength. In general, shorter sequences will give better signal to noiseratios than longer probes and will therefore be more sensitive. However,in certain embodiments, shorter probes might not be the best substratefor the nuclease, so some degree of empiric optimization for length isneeded. In certain embodiments, the oligonucleotide comprises 0-50%purines (or any value in between). In certain embodiments theoligonucleotide comprises 100% pyrimidines.

It should be noted that the specific sequence of the oligonucleotide isnot critical. Certain combinations of purines and pyrimidines aresusceptible to bacterial endonucleases, while resisting mammaliannucleases. Endonucleases are enzymes that cleave the phosphodiester bondwithin a polynucleotide chain, in contrast to exonucleases, which cleavephosphodiester bonds at the end of a polynucleotide chain. Thesebacterial nucleases are not sequence-specific like restriction enzymes,which typically require a recognition site and a cleavage pattern. Someendonucleases cleave single-stranded nucleic acid molecules, whileothers cleave double-stranded nucleic acid molecules. For example, thedata below show a time-course of activity of the Mycoplasma-derivednuclease and demonstrate that the Mycoplasma nuclease can digest avariety of distinct sequences. The earliest time-point shows partialdegradation of the 51 nt long sequence modified with either 2′-fluoro or2′-O-methyl pyrimidines, with intermediate degradation products clearlyvisible. Each of the degradation products of intermediate size is infact a distinct substrate and these are clearly being digested as seenin the later time points.

Fluorophores

In certain embodiments, the oligonucleotides of the present inventionare operably linked to one or more fluorophores, which may also becalled a “fluorescent tag.” A fluorophore is a molecule that absorbslight (i.e. excites) at a characteristic wavelength and emits light(i.e. fluoresces) at a second lower-energy wavelength. Fluorescencereporter groups that can be incorporated into Substrate compositionsinclude, but are not limited to, fluorescein, tetrachlorofluorescein,hexachlorofluorescein, tetramethylrhodamine, rhodamine,cyanine-derivative dyes, Texas Red, Bodipy, and Alexa dyes.Characteristic absorption and emission wavelengths for each of these arewell known to those of skill in the art.

A fluorescence quencher is a molecule that absorbs or releases energyfrom an excited fluorophore (i.e., reporter), returning the fluorophoreto a lower energy state without fluorescence emission at the wavelengthcharacteristic of that fluorophore. For quenching to occur, reporter andquencher must be in physical proximity. When reporter and quencher areseparated, energy absorbed by the reporter is no longer transferred tothe quencher and is instead emitted as light at the wavelengthcharacteristic of the reporter. Appearance of a fluorescent signal fromthe reporter group following removal of quenching is a detectable eventand constitutes a “positive signal” in the assay of the presentinvention, and indicates the presence of RNase in a sample.

Fluorescence quencher groups include molecules that do not emit anyfluorescence signal (“dark quenchers”) as well as molecules that arethemselves fluorophores (“fluorescent quenchers”). Substratecompositions that employ a “fluorescent quencher” will emit light bothin the intact and cleaved states. In the intact state, energy capturedby the reporter is transferred to the quencher via FRET and is emittedas light at a wavelength characteristic for the fluorescent quencher. Inthe cleaved state, energy captured by the reporter is emitted as lightat a wavelength characteristic for the reporter. When compositions thatemploy fluorescent quenchers are used in a FRET assay, detection must bedone using a fluorometer. In certain embodiments, Substrate compositionsthat employ a “dark quencher” will emit light only in the cleaved state,enabling signal detection to be performed visually (detection may alsobe done using a fluorometer). Visual detection is rapid, convenient, anddoes not require the availability of any specialized equipment. It isdesirable for an RNase detection assay to have visual detection methodas an available option. Substrate compositions employing a “darkquencher” enable a visual detection endonuclease assay while Substratecompositions employing a “fluorescent quencher” are incompatible with avisual detection assay.

In one embodiment of the invention, the Substrate is comprised of afluorescence quencher group that does not itself emit a fluorescencesignal, i.e. is a “dark quencher”. “Dark quenchers” useful incompositions of the invention include, but are not limited to, dabcyl,QSY™-7, QSY-33 (4′,5-dinitrofluorescein, pipecolic acid amide) andBlack-Hole Quenchers™ 1, 2, and 3 (Biosearch Technologies, Novato,Calif.). Assay results (i.e., signal from cleaved Substrate) can thus bedetected visually. Optionally, the fluorescence signal can be detectedusing a fluorometer or any other device capable of detecting fluorescentlight emission in a quantitative or qualitative fashion.

In certain embodiments, the fluorophore is one or more of thefluorophores listed in Table 1.

TABLE 1 Excitation Emission Probe (nm) (nm) Hydroxycoumarin 325 386Alexa fluor 325 442 Aminocoumarin 350 445 Methoxycoumarin 360 410Cascade Blue (375); 401   423 Pacific Blue 403 455 Pacific Orange 403551 Lucifer yellow 425 528 Alexa fluor 430 430 545 NBD 466 539R-Phycoerythrin (PE) 480; 565 578 PE-Cy5 conjugates 480; 565; 650 670PE-Cy7 conjugates 480; 565; 743 767 Red 613 480; 565 613 PerCP 490 675Cy2 490 510 TruRed 490, 675 695 FluorX 494 520 Fluorescein 495 519 FAM495 515 BODIPY-FL 503 512 TET 526 540 Alexa fluor 532 530 555 HEX 535555 TRITC 547 572 Cy3 550 570 TMR 555 575 Alexa fluor 546 556 573 Alexafluor 555 556 573 Tamara 565 580 X-Rhodamine 570 576 Lissamine RhodamineB 570 590 ROX 575 605 Alexa fluor 568 578 603 Cy3.5 581 581 596 TexasRed 589 615 Alexa fluor 594 590 617 Alexa fluor 633 621 639 LC red 640625 640 Allophycocyanin (APC) 650 660 Alexa fluor 633 650 688 APC-Cy7conjugates 650; 755 767 Cy5 650 670 Alexa fluor 660 663 690 Cy5.5 675694 LC red 705 680 710 Alexa fluor 680 679 702 Cy7 743 770 IRDye 800 CW774 789

In certain in vivo embodiments, the fluorophore emits in the nearinfrared range, such as in the 650-900 nm range. (Weissleder et al.,“Shedding light onto live molecular targets, Nature Medicine, 9:123-128(2003)).

Fluorescence Quencher Group

In certain embodiments, the oligonucleotides of the present inventionare operably linked to one or more fluorescence quencher group or“quencher.”

In certain embodiments, the quencher is one or more of the quencherslisted in Table 2.

TABLE 2 Absorption Maximum Quencher (nm) DDQ-I 430 Dabcyl 475 Eclipse530 Iowa Black FQ 532 BHQ-1 534 QSY-7 571 BHQ-2 580 DDQ-II 630 IowaBlack RQ 645 QSY-21 660 BHQ-3 670 IRDye QC-1 737 ZEN 532

Additional quenchers are described in U.S. Pat. No. 7,439,341, which isincorporated by reference herein.

Linkers

In certain embodiments, the oligonucleotide is linked to the fluorophoreand/or quencher by means of a linker.

In certain embodiments, an aliphatic or ethylene glycol linker (as arewell known to those will skill in the art) is used. In certainembodiments, the linker is a phosphodiester linkage. In certainembodiments, the linker is a phosphorothioate linkage. In certainembodiments, other modified linkages between the modifier groups likedyes and quencher and the bases are used in order to make these linkagesmore stabile, thereby limiting degradation to the nucleases.

In certain embodiments, the linker is a binding pair. In certainembodiments, the “binding pair” refers to two molecules which interactwith each other through any of a variety of molecular forces including,for example, ionic, covalent, hydrophobic, van der Waals, and hydrogenbonding, so that the pair have the property of binding specifically toeach other. Specific binding means that the binding pair members exhibitbinding to each other under conditions where they do not bind to anothermolecule. Examples of binding pairs are biotin-avidin, hormone-receptor,receptor-ligand, enzyme-substrate, IgG-protein A, antigen-antibody, andthe like. In certain embodiments, a first member of the binding paircomprises avidin or streptavidin and a second member of the binding paircomprises biotin.

In certain embodiments, the oligonucleotide is linked to the fluorophoreand/or quencher by means of a covalent bond.

In certain embodiments, the oligonucleotide probe, i.e., anoligonucleotide that is operably linked to a fluorophore and quencher,is also operably linked to a solid substrate. For example, theoligonucleotide probe may be linked to a magnetic bead.

Chemistries that can be used to link the fluorophores and quencher tothe oligonucleotide are known in the art, such as disulfide linkages,amino linkages, covalent linkages, etc. In certain embodiments,aliphatic or ethylene glycol linkers that are well known to those withskill in the art can be used. In certain embodiments, phosphodiester,phosphorothioate and/or other modified linkages between the modifiergroups like dyes and quencher are used. These linkages provide stabilityto the probes, thereby limiting degradation to nucleobases. Additionallinkages and modifications can be found on the world-wide-web attrilinkbiotech.com/products/oligo/oligo_modifications.asp.

Detection Compositions

In certain embodiments, the probes described above can be prepared aspharmaceutically-acceptable compositions. In certain embodiments, theprobes are administered so as to result in the detection of a microbialinfection. The amount administered will vary depending on variousfactors including, but not limited to, the composition chosen, theparticular disease, the weight, the physical condition, and the age ofthe mammal. Such factors can be readily determined by the clinicianemploying animal models or other test systems, which are well known tothe art.

Pharmaceutical formulations, dosages and routes of administration fornucleic acids are generally known in the art. The present inventionenvisions detecting a microbial infection in a mammal by theadministration of a probe of the invention. Both local and systemicadministration is contemplated.

One or more suitable unit dosage forms of the probe of the invention canbe administered by a variety of routes including parenteral, includingby intravenous and intramuscular routes, as well as by direct injectioninto the diseased tissue. The formulations may, where appropriate, beconveniently presented in discrete unit dosage forms and may be preparedby any of the methods well known to pharmacy. Such methods may includethe step of bringing into association the probe with liquid carriers,solid matrices, semi-solid carriers, finely divided solid carriers orcombinations thereof, and then, if necessary, introducing or shaping theproduct into the desired delivery system.

When the probes of the invention are prepared for administration, incertain embodiments they are combined with a pharmaceutically acceptablecarrier, diluent or excipient to form a pharmaceutical formulation, orunit dosage form. The total active ingredient (i.e., probe) in suchformulations include from 0.1 to 99.9% by weight of the formulation. A“pharmaceutically acceptable” is a carrier, diluent, excipient, and/orsalt that is compatible with the other ingredients of the formulation,and not deleterious to the recipient thereof. The active ingredient foradministration may be present as a powder or as granules, as a solution,a suspension or an emulsion.

Pharmaceutical formulations containing the probe of the invention can beprepared by procedures known in the art using well known and readilyavailable ingredients. The therapeutic agents of the invention can alsobe formulated as solutions appropriate for parenteral administration,for instance by intramuscular, subcutaneous or intravenous routes.

The pharmaceutical formulations of probe of the invention can also takethe form of an aqueous or anhydrous solution or dispersion, oralternatively the form of an emulsion or suspension.

Thus, probe may be formulated for parenteral administration (e.g., byinjection, for example, bolus injection or continuous infusion) and maybe presented in unit dose form in ampules, pre-filled syringes, smallvolume infusion containers or in multi-dose containers with an addedpreservative. The probe may take such forms as suspensions, solutions,or emulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.Alternatively, the probe may be in powder form, obtained by asepticisolation of sterile solid or by lyophilization from solution, forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water,before use.

The pharmaceutical formulations of the present invention may include, asoptional ingredients, pharmaceutically acceptable carriers, diluents,solubilizing or emulsifying agents, and salts of the type that arewell-known in the art. Specific non-limiting examples of the carriersand/or diluents that are useful in the pharmaceutical formulations ofthe present invention include water and physiologically acceptablebuffered saline solutions such as phosphate buffered saline solutions pH7.0-8.0, saline solutions, and water.

Substrate Synthesis

Synthesis of the nucleic acid Substrate of the invention can beperformed using solid-phase phosphoramidite chemistry (U.S. Pat. No.6,773,885) with automated synthesizers, although other methods ofnucleic acid synthesis (e.g., the H-phosphonate method) may be used.Chemical synthesis of nucleic acids allows for the production of variousforms of the nucleic acids with modified linkages, chimericcompositions, and nonstandard bases or modifying groups attached inchosen places throughout the nucleic acid's entire length.

Detectable Bacteria

The following bacteria can be detected using the methods of the presentinvention:

Staphylococcus aureus

Staphylococcus epidermidis

Staphylococcus lugdunensis

Staphylococcus saprophyticus

Streptococcus pyogenes

Streptococcus agalactiae

Streptococcus pneumoniae

Streptococcus mutans

Listeria monocytogenes

Corynebacterium diphtheriae

Bordetella pertussis

Clostridium difficile

Clostridium perfringens

Clostridium botulinum

Enterobacter cloacae

Citrobacter freundii

Borrelia burgdorferi

Treponema pallidum

Bacillus anthracis

Bacillus cereus

Enterococcus faecalis

Enterococcus faecium

Pseudomonas aeruginosa

Acinetobacter baumannii

Yersinia pestis

Yersinia pseudotuberculosis

Yersinia enterocolitica

Klebsiella pneumoniae

Vibrio cholerae

Salmonella enterica

Salmonella typhi

Escherichia coli

Neisseria gonorrhoeae

Neisseria meningitidis

Mycobacterium tuberculosis

Haemophilus influenzae

Legionella pneumophila

Francisella tularensis

Bacteroides fragilis

Brucella abortus

Mycoplasma fermentans

Mycoplasma pneumoniae

Mycoplasma genitalium

Chlamydia trachomatis

Detectable Viruses

In addition to the bacterial pathogens listed above, the presentinvention can also detect human Cytomegalovirus (CMV, also known asHuman Herpes Virus 5). It also detects related viruses which includeHuman Herpes Viruses 1, 2, 3, 4, 5, 6A, 6B, 7 and 8.

Detection Methods

In certain embodiments, the present invention provides methods fordetecting bacteria in a sample in vitro or in vivo. The method of theinvention proceeds in the following steps: combine “test sample” withSubstrate(s) to produce a mixture, the mixture being the Assay Mix,incubate, and detect fluorescence signal. “Test sample” refers to anymaterial being assayed for endonuclease (e.g., ribonuclease) activityand in certain embodiments, will be a liquid. Solids can be indirectlytested for the presence of RNase contamination by washing or immersionin solvent, e.g., water, followed by assay of the solvent.

For example, one can contact a sample with an oligonucleotide probe asdescribed herein, and detect the presence of bacterial endonucleasesusing a florometer. Alternatively, oligonucleotide probes orcompositions can be administered in vivo to a patient (e.g. injected insitu into a mammal) and fluorescence in the organism can be measured. Incertain embodiments, the in vivo fluorescence can be measured to a depthof about 7-14 cm. Thus, in certain embodiments, the probes of thepresent invention can be use in medical diagnostic applications andmedical diagnostic imaging.

In certain embodiments, the probes of the present invention are alsouseful to detect bacterial contamination in settings such as researchlaboratories.

Assay Mix.

The Substrate is mixed and incubated with the test sample. This mixtureconstitutes the Assay Mix. Ideally, the Assay Mix is a small volume,from about 1 μl to about 10 mls, or, from about 10 to 100 μl. Theprecise volume of the Assay Mix will vary with the nature of the testsample and the detection method. Optionally, a buffer can be added tothe Assay Mix. Nucleases, including some ribonucleases, require thepresence of divalent cations for maximum activity and providing anoptimized buffered solution can increase the reaction rate and therebyincrease assay sensitivity. Buffers of different composition can beused, as described in U.S. Pat. No. 6,773,885. In certain embodiments,control reactions are included, but are not essential. A NegativeControl Mix, for example, comprises a solution of Substrate in water orbuffer without any test sample or added nuclease. In this control, theSubstrate should remain intact (i.e., without fluorescence emission). Ifthe Negative Control Mix results in positive signal, then the quality ofall reagents is suspect and fresh reagents should be employed. Possiblecauses of a signal in a Negative Control include degradation of theSubstrate or contamination of any component reagent with endonuclease(e.g., ribonuclease) activity. A Positive Control Mix, for example,comprises a solution of Substrate in water or buffer plus a known,active RNase enzyme. If the Positive Control Mix results in a negativesignal, then the quality of all reagents is suspect and fresh reagentsshould be employed. Possible causes of a negative Positive Control Mixinclude defective Substrate or contamination of any component reagentwith an endonuclease (e.g., a ribonuclease) inhibitor. Any RNase thatcleaves the Substrate can be employed for use in the Positive ControlMix. In one embodiment, RNase A is used, as this enzyme is bothinexpensive and readily available. Alternatively, RNase 1 can be used.RNase 1 is heat labile and is more readily decontaminated fromlaboratory surfaces.

Incubation.

The Assay Mix (e.g., the test sample plus Substrate) is incubated.Incubation time and condition can vary from a few minutes to 24 hours orlonger depending upon the sensitivity required. Incubation times of onehour or less are desirable. Endonucleases (e.g., ribonucleases) arecatalytic. Increasing incubation time should therefore increasesensitivity of the Assay, provided that background cleavage of theSubstrate (hydrolysis) remains low. As is evident, assay background isstable over time and Assay sensitivity increases with time ofincubation. Incubation temperature can generally vary from roomtemperature to 37.degree. C. but may be adjusted to the temperatureoptimum of a specific endonuclease (e.g., ribonuclease) suspected asbeing present as a contaminant.

Signal Detection.

Fluorescence emission can be detected using a number of techniques (U.S.Pat. No. 6,773,885). In one method of detection, visual inspection isutilized. Visual detection is rapid, simple, and can be done withoutneed of any specialized equipment. Alternatively, detection can be doneusing fluorometry or any other method that allows for qualitative orquantitative assessment of fluorescent emission.

Visual Detection Method.

Following incubation, the Assay Mix is exposed to UV light to provideexcitation of the fluorescence reporter group. An Assay Mix in which theSubstrate remains intact will not emit fluorescent signal and willvisually appear clear or dark. Absence of fluorescence signalconstitutes a negative assay result. An Assay Mix in which the Substratehas been cleaved will emit fluorescent signal and will visually appearbright. Presence of fluorescence signal constitutes a positive assayresult, and indicates the presence of RNase activity in the sample. Thevisual detection method is primarily intended for use as a qualitativeendonuclease (e.g., ribonuclease) assay, with results being simplyeither “positive” or “negative”. However, the assay is crudelyquantitative in that a bright fluorescent signal indicates higher levelsof RNase contamination than a weak fluorescent signal.

The Assay Mix will ideally constitute a relatively small volume, forexample 10 to 100 μl, although greater or lesser volumes can beemployed. Small volumes allow for maintaining high concentrations ofSubstrate yet conserves use of Substrate. The visual detection Assay inone embodiment uses 50 pmoles of Substrate at a concentration of 0.5 μMin a 100 μl final volume Assay Mix. Lower concentration of Substrate(e.g., below 0.1 uM) will decrease assay sensitivity. Higherconcentrations of Substrate (e.g., above 1 μM) will increase backgroundand will unnecessarily consume Substrate.

Steps (mixing, incubating, detecting), can be performed in one tube. Inone embodiment, the tube is a small, thin-walled, UV transparentmicrofuge tube, although tubes of other configuration may be used. A“short wave” UV light source emitting at or around 254 nm is used in oneembodiment for fluorescence excitation. A “long wave” UV light sourceemitting at or around 300 nm can also be employed. A high intensity,short wave UV light source will provide for best sensitivity. UV lightsources of this kind are commonly found in most molecular biologylaboratories. Visual detection can be performed at the laboratory benchor in the field, however sensitivity will be improved if done in thedark.

Fluorometric Detection Method.

Following incubation fluorescence emission can be detected using afluorometer. Fluorometric detection equipment includes, but is notlimited to, single sample cuvette devices and multiwell plate readers.As before, mixing, incubation, and detection can be performed in thesame vessel. Use of a multiwell plate format allows for small samplevolumes, such as 200 μl or less, and high-throughput robotic processingof many samples at once. This format is used in certain industrial QCsettings. The method also provides for the Assay to be performed inRNase free cuvettes. As before, mixing, incubation, and detection can beperformed in the same vessel. Use of fluorometric detection allows forhighly sensitive and quantitative detection.

Kits

The present invention further includes kits for detecting endonuclease(e.g., ribonuclease) activity in a sample, comprising Substrate nucleicacid(s) and instructions for use. Such kits may optionally contain oneor more of: a positive control endonuclease (e.g., ribonuclease),RNase-free water, a buffer, and other reagents. The kits may includeRNase-free laboratory plasticware, such as thin-walled, UV transparentmicrotubes and/or multiwell plates for use with the visual detectionmethod and multiwell plates for use with plate-fluorometer detectionmethods.

One kit of the invention includes a universal Substrate, the Substratebeing sensitive to a broad spectrum of endonuclease (e.g., ribonuclease)activity. The kit is intended to detect endonuclease (e.g.,ribonuclease) activity from a variety of sources. The assay iscompatible with visual detection. In certain embodiments, the Substratewill be provided in dry form in individual thin-walled, UV transparentmicrotubes, or in multiwell (e.g., 96 well) formats suitable for highthroughput procedures. Lyophilized Substrate has improved long-termstability compared to liquid solution in water or buffer. If provided inliquid solution, stability is improved with storage at least below −20°C., such as at −80° C. Storage in individual aliquots limits potentialfor contamination with environmental endonuclease (e.g., ribonucleases).Alternatively, the Substrate can be provided in bulk, either lyophilizedor in liquid solution. Alternatively, substrate can be provided in bulkand can be dispersed at the discretion of the user.

An additional kit of the invention includes a set of enzyme-specific orenzyme-selective Substrates that together detect most RNase activitiesand individually can be used to distinguish between differentendonuclease (e.g., ribonuclease) enzymes. Such a kit can be used toassess the nature and source of RNase contamination or can measureactivity of specific enzyme of interest.

In Vitro Assays for Evaluating Nuclease Activity

In certain embodiments, the present invention provides in vitro assaysfor evaluating the activity of microbial nucleases on various nucleicacid substrates. In certain embodiments the assay evaluates the activityof Mycoplasma nucleases. In certain embodiments the assay evaluates theactivity of bacterial (e.g., Staphylococcus aureus or Streptococcuspneumonia) or viral nucleases. For example, a biological sample (e.g.,tissue, cells, biological fluids) or material derived from such a sampleis combined with an oligonucleotide-based probe and incubated for aperiod to time. The fluorescence level of this reaction is then measured(e.g., with a fluorometer), and compared with the fluorescence levels ofsimilar reactions that serve as positive and negative controls.

Selective Inactivation of Serum Nucleases in Serum and Plasma SamplesContaining Micrococcal Nuclease.

To selectively inactivate mammalian (mouse or human) serum nucleases,while preserving the activity of micrococcal nuclease, we have developeda simple heat-based protocol that denatures and inactivates themammalian nucleases. Micrococcal nuclease is known to re-fold into itsnatural conformation after heat-denaturation in buffers containingsufficient concentrations of calcium and proteins. The survival of serumnucleases subjected to these same conditions has not, to the best of ourknowledge, been studied. After dialyzing mouse or human serum (or humanplasma) that was spiked with varying concentrations of micrococcalnuclease against a buffer of 50 mM Tris-HCl, pH 9.0, 10 mM CaCl2, thesamples were incubated at 90° C. for 20 minutes. The samples were thenallowed to cool to room temperature, centrifuged in a microcentrifuge topellet the proteins that had aggregated and the supernatants weretransferred to fresh tubes. 9 microliters of each supernatant was thencombined with 50 picomoles of the Poly TT Probe in a 10 microliterreaction and incubated at 37° C. for 1 hour. Each reaction was thendiluted in a “stop” buffer (290 uL of 10 mM EDTA+10 mM EGTA in PBS) anddivided into 3 portions for triplicate plate-reader fluorescencemeasurements. The nuclease activity of serum samples in whichmicrococcal nuclease was not added was close to background levels (i.e.,levels observed when probe is incubated with buffer only). The samplesin which even very small amounts of micrococcal nuclease were addedexhibited strong nuclease activity against this probe after thisprotocol was carried out. This method may be useful in detecting thepresence of low concentrations of micrococcal nuclease in clinicalspecimens such as blood serum.

Staphylococcus aureus Detection Method

In certain embodiments, the present invention provides a method ofdetecting Staphylococcus aureus in a test sample, comprising:

(a) contacting the test sample with a probe of any one of claims 1-xxxto form a digested probe,

(b) collecting the digested probe, and

(c) measuring the fluorescence emitted by the digested probe.

In certain embodiments, the test sample is a biological sample. Incertain embodiments, the biological sample is a blood sample. In certainembodiments, the blood sample is whole blood, serum or plasma.

In certain embodiments, the sample further comprises calcium chloride.In certain embodiments, the calcium chloride is at a concentration ofabout 5 to 20 mM. In certain embodiments, the calcium chloride is at aconcentration of about 10 mM.

In certain embodiments, the sample has been heated at 55-100° C. for 10seconds to 20 hours to form a heat-treated test sample prior to testing.In certain embodiments, the sample has been heated at about 70 to 95° C.In certain embodiments, the sample has been heated for about 15-30minutes. In certain embodiments, the sample has been heated at about 90°C. for about 20 minutes to form a heat-treated test sample prior totesting.

In certain embodiments, the sample has been clarified after the heatingstep. In certain embodiments, the clarification is by means ofcentrifugation at 1 k to 20 k×g for 10 seconds to 20 minutes after theheating step to form a heat-treated, clarified supernatant test sample.In certain embodiments, the clarification is by means of centrifugationat about 17 k×g for about 10 minutes after the heating step to form aheat-treated, clarified supernatant test sample. In certain embodiments,the clarification is by means of filtration after the heating step toform a heat-treated, clarified supernatant test sample.

In certain embodiments, the heat-treated test sample has beenconcentrated prior to testing. In certain embodiments, the concentrationis by means of immunoprecipitation. In certain embodiments, theimmunoprecipitation is by means of anti-micrococcal nucleaseantibody-coupled magnetic beads. In certain embodiments, the magneticbeads are Protein G-coupled magnetic beads.

In certain embodiments, the probe is the probe isFAM/TTTTTTTTTTT/ZEN/IAbRQSp/(SEQ ID NO. 5), wherein 6-FAM is afluorescein amidite fluorophore, ZEN is a ZEN dark quencher, and IAbRQSpis a Iowa Black dark quencher, and the fluorescence is measured at485/530 nm excitation/emission.

In certain embodiments, the present invention provides a Staphylococcusaureus detection method. In certain embodiments, the method involves thefollowing steps:

-   -   1. Add CaCl₂ to plasma for a final concentration of ˜10 mM.    -   2. Heat plasma to 90° C. for 20 minutes.    -   3. Centrifuge heat precipitated plasma at 17,000×g for 10 min.    -   4. Resuspend anti-micrococcal nuclease antibody-coupled magnetic        beads with heat-treated plasma supernatant.    -   5. Wash antibody-nuclease-bead complex.    -   6. Incubate PolyTT probe in optimal buffer with        nuclease-antibody-bead complex.    -   7. Collect digested probe in buffer from nuclease-antibody-bead        complex.    -   8. Measure fluorescence.

Example 1

Because of the difficulty in detecting trace quantities of E. colirapidly with field-compatible methods, the present invention wasdeveloped. The invention is a pair of self-hybridizing, quenchedfluorescent oligonucleotide probes that are digested (i.e., cleaved) andthereby activated by Endonuclease I, a deoxyribonuclease (DNase)expressed in Escherichia coli (E. coli). The probes enable the detectionof as few as 219 E. coli bacterial cells after brief (as little as 1hour) incubations.

Probes

The invention consists of a pair of quenched fluorescent, chemicallymodified oligonucleotide probes. These synthetic molecules have afluorophore on one end and a pair of quenching moieties on the otherend. The quenchers greatly diminish the fluorescence of the fluorophore,due to their physical properties and their close proximity. Upondegradation of the oligonucleotide, the quenchers diffuse away from thefluorophore and the fluorophore then exhibits much greater fluorescence.

In one embodiment, the probes were as follows:

(SEQ ID NO: 27) Oligo 1 (8-mer DNA): /56-FAM/CTACGTAG/ZEN// 3IAbRQSp/where 56-FAM is a FAM fluorophore (fluorescein amidite), the boldletters indicate deoxy nucleotides (DNA), ZEN is the ZEN fluorescencequencher and 3IAbRQSp is the Iowa Black fluorescence quencher. The probeis listed from the 5′- to the 3′-ends.

(SEQ ID NO: 4) Self-Hyb Fl: /56-FAM/fCfUfAfCfGfUfAfG/ZEN// 3IAbRQSp/where 56-FAM is a FAM fluorophore (fluorescein amidite), nucleotides arefA (2′-fluoro modified A), fC (2′-fluoro modified C), fG (2′-fluoromodified G) and fU (2′-fluoro modified U), ZEN is the ZEN fluorescencequencher and 3IAbRQSp is the Iowa Black fluorescence quencher. The probeis listed from the 5′- to the 3′-ends.

The probes are also self-complementary, meaning that each will bind anidentical copy of itself, oriented in the opposite direction, forming adouble-stranded nucleic acid substrate for nucleases. In thisdouble-stranded form, the probes serve as robust substrates forEndonuclease I, a nuclease expressed in E. coli. that prefersdouble-stranded DNA substrates. When the probes are incubated in lysatesof E. coli cells, the Endonuclease I which is present in the lysatesdegrades them, resulting in an increase in fluorescence. The followingoptimal buffer was developed for these reactions: 50 mM Tris-HCl, pH8.0, 100 mM NaCl, 12 mM MgCl₂, 1% Triton X-100, 1 mM DTT, 1× ProteaseInhibitor Cocktail (cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpackfrom Roche, cat #05 892 791 001; 1× is 1 tablet per 10 ml as specifiedin product literature).

For detection of E. coli in the field, the probes would need to becoupled with a field-compatible bacterial concentration device and witha field-compatible fluorescence measuring device.

Results

The inventors previously developed quenched fluorescent,nuclease-activated oligonucleotide probes that detect the presence ofStaphylococcus aureus by detecting the activity of one of its nucleases.These probes have a fluorophore on their 5′-ends that exhibits verylittle fluorescence because tow fluorescence quenchers, which arecoupled to the 3′-end, are in close proximity. Upon digestion of theoligonucleotide portion, the quenchers diffuse away from thefluorophore, resulting in its unquenching and thus activation of theprobe. With the present invention, the inventors explored whetherEscherichia coli might also be detected in a similar manner.Endonuclease 1 of E. coli is commonly deleted in strains used formolecular biology procedures in order to increase the yield of plasmidDNA production (Taylor, R. G., Walker, D. C. & Mcinnes, R. R.Escherichia-Coli Host Strains Significantly Affect the Quality ofSmall-Scale Plasmid DNA Preparations Used for Sequencing. Nucleic AcidsRes 21, 1677-1678 (1993)). This protein, therefore, was focused on as acandidate enzyme of E. coli that might be used for its detection.

A DNA zymogram was performed (FIG. 1) with E. coli lysates to measure E.coli nuclease activities. In addition to the FDA strain Seattle 1946 (arepresentative coliform E. coli strain), the inventors also included anEndonuclease 1 knockout strain (Keio EndA KO) of the Keio collection andits parental strain (K12) as a control in this experiment (Baba, T., etal. Construction of Escherichia coli K-12 in-frame, single-gene knockoutmutants: the Keio collection. Mol Syst Biol 2, 2006 0008 (2006)). In thezymogram assay, dark bands indicate digestion of the DNA that isembedded in the gel and thus nuclease activity of the resolved proteins.A roughly 25 kDa band is clearly evident in the coliform E. coli lysateand a band of the same size is also present in the Keio parental E. colistrain, while no band of the corresponding size is visible in the EndAstrain that lacks Endonuclease 1. Western blot analysis of Endonuclease1 in lysates of this and another Keio collection Endonuclease 1 knockoutE. coli strain (FIG. 2) revealed absence of bands of the approximatemolecular weight of Endonuclease 1, thus confirming the Endonuclease1-null status of these strains. Together, these data support the notionthat Endonuclease 1 is a viable candidate for detecting coliform E.coli.

Next, Endonuclease 1 activity was measured with a quenched fluorescentprobe rather than a gel-based assay due to the simplicity andfield-compatibility of this simpler approach (Kelemen, B. R., et al.Hypersensitive substrate for ribonucleases. Nucleic Acids Res 27,3696-3701 (1999)). A single-stranded DNA probe was not activated (i.e.,signal did not exceed that of the buffer only control) in lysates of thecoliform E. coli strain, the Keio collection parental strain or any ofthe Keio collection mutants tested (FIG. 3). When the same probe wastested after being made into a double-stranded form by annealing anunlabeled complementary DNA oligonucleotide (see Table 3 for probedetails), the probe was activated (i.e., signal exceeded that in thebuffer only control) in several of the lysates tested. In particular,the lysates of the coliform E. coli, the Keio parental strain and thenfi and nth Keio collection mutants (these have different nucleasesdeleted) all produced probe activation that exceeded that seen in thebuffer only control (Asahara, H., Wistort, P. M., Bank, J. F., Bakerian,R. H. & Cunningham, R. P. Purification and characterization ofEscherichia coli endonuclease III from the cloned nth gene. Biochemistry28, 4444-4449 (1989); Guo, G., Ding, Y. & Weiss, B. nfi, the gene forendonuclease V in Escherichia coli K-12. J Bacteriol 179, 310-316(1997)).

TABLE 3 Names, sequences and modifications ofoligonucleotide probes used Oligo SEQ ID Name Oligo Sequence NO 4xAFAM-mUmCmUmCAAAAmGmUmAmC- 28 DNA ZEN-RQ 4xT mGmUmAmCTTTTmGmAmGmA 29 CompSelf-Hybridizing Probes Oligo 1 FAM-CTACGTAG-ZEN-RQ SEQ ID NO: 30Self-Hyb FAM- + C + T + A + C + G + SEQ ID NO: 31 LNA T + A + G-ZEN-RQSelf-Hyb FAM-mCmUmAmCmGmUmAmG-ZEN-RQ SEQ ID NO: 32 OMe Self-HybFAM-fCfUfAfCfGfUfAfG-ZEN-RQ SEQ ID NO: 33 Fl FAM FAM fluorophore(fluorescein amidite) ZEN “ZEN” fluorescence quencher RQ “Iowa Black”fluorescence quencher mA 2′-O-methyl modified A mC 2′-O-methyl modifiedC mG 2′-O-methyl modified G mU 2′-O-methyl modified U fA 2′-fluoromodified A fC 2′-fluoro modified C fG 2′-fluoro modified G fU 2′-fluoromodified U +A Locked nucleic acid modified A +C Locked nucleic acidmodified C +G Locked nucleic acid modified G +U Locked nucleic acidmodified U Nucleotides written in bold are deoxy nucleotides (DNA). Allsequences are written from 5′ to 3′ orientation.

It was noted that the signal of the double-stranded fluorescent probeincubated in buffer was substantially greater than the same probe in itssingle-stranded form incubated in buffer. This is likely due to therigid helical structure of the double-stranded form which forces thefluorophore apart from the quenchers. In contrast, the flexibility ofthe single-stranded form likely allows for hydrophobic interactionsbetween the fluorophore and quenchers that will promote substantiallygreater quenching. Neither of the lysates of 2 distinct Endonuclease 1knockout strains from the Keio collection (6023 and 8144) activated thedouble-stranded probe above the level seen in the buffer only control.The activity seen in the other lysates can therefore be attributed toEndonuclease 1. That Endonuclease 1 has a preference for double-strandedDNA is consistent with previous published studies (Lehman, I. R.,Roussos, G. G. & Pratt, E. A. The deoxyribonucleases of Escherichiacoli. II. Purification and properties of a ribonucleic acid-inhibitableendonuclease. J Biol Chem 237, 819-828 (1962)).

Next, the inventors developed a quenched fluorescent oligonucleotideprobe that is highly sensitive to Endonuclease 1, and also exhibits alow basal level of fluorescence (i.e., strong quenching). Aself-annealing probe configuration (see FIG. 4 for cartoonrepresentation) yields a double-stranded substrate for enzyme digestionand also fixes the quenchers of one probe in close proximity to thefluorophore of its binding partner for strong quenching. A DNA versionof this probe (Oligo 1) was efficiently digested in lysates of thecoliform E. coli strain (FIGS. 5-9).

To identify the optimal buffer conditions for digestion of Oligo 1 incoliform E. coli lysates, the inventors compared the activation of thisprobe in buffers containing different divalent cations (FIG. 5), variouspHs (FIG. 5), various divalent cation concentrations (FIG. 6) andvarious sodium chloride concentrations (FIG. 7). These results yieldedan optimized Endonuclease 1 reaction buffer consisting of 50 mMTris-HCl, pH 8, 1% Triton X-100, 1 mM DTT, 1× protease inhibitors, 12 mMMgCl₂, 100 mM NaCl (see Methods for details regarding the proteaseinhibitors). Subsequent experiments were carried out with this optimizedbuffer.

To determine whether the digestion of Oligo 1 in the optimized bufferwas indeed due to Endonuclease 1 activity, we measured Oligo 1activation in lysates of coliform E. coli and the Keio collectionparental, and Endonuclease 1, nfi and nth knockout strains (FIG. 8).Activation of the probe in all the lysates except those of theEndonuclease 1 knockout strains confirms that Endonuclease 1 is thenuclease responsible for Oligo 1 digestion in the lysates in which it ispresent.

Next, the activation of Oligo 1 was compared to that of three additionalprobes that are identical, except that the nucleotides are modifieddifferently (see Table 3 for a complete description of the probes). Themodifications were chosen based on those that are known to provideresistance to many nucleases, such as mammalian serum nucleases (Behlke,M. A. Chemical modification of siRNAs for in vivo use. Oligonucleotides18, 305-319 (2008)). One of these additional probes, “Self-Hyb Fl”, inwhich 2′-fluoro modified RNA nucleotides were substituted for the DNAnucleotides of Oligo 1, was robustly activated in the coliform E. colilysate (FIG. 9). To determine whether activation of the Self-Hyb Flprobe is also due to Endonuclease 1, we again used the Keio collectionparental and nuclease knockout strains in a probe activation experiment(FIG. 10). As with Oligo 1 probe activation, the Self-Hyb Fl probe isselectively activated in the lysates in which Endonuclease 1 is present,thus indicating that Endonuclease 1 is also the nuclease responsible forits activation.

Finally, to determine the minimal number of E. coli bacterial cells thatcan be detected with this approach in its present form, the activationof the Self-Hyb Fl probe was measured in various dilutions of a lysateof coliform E. coli cells (FIG. 11). Using the optical density of theculture (measured prior to lysis) to estimate the concentration ofbacteria, it was found that the equivalent of as few as 219 bacterialcells per well of the plate-reader can be detected above the backgroundlevel measured with buffer only.

In summary, nuclease-activated oligonucleotide probes were developedthat can rapidly detect the presence of E. coli with high sensitivity.

Methods

Culture Growth and Processing

Overnight cultures of indicated E. coli strains were grown at 37° C. ina shaking incubator. Coliform E. coli was grown in Tryptic soy brothwithout antibiotics, the Keio collection mutants were grown in LBsupplemented with kanamycin. The Keio parental K12 strain was grown inLB without antibiotics. Bacteria from 1 milliliter of each culture waspelleted by centrifugation, washed in 1 ml of 10 mM Tris-HCl pH 7.4 andlysed with 30 mM Tris-HCl pH8.0, 1 mM EDTA, 20% sucrose, 10 μg/mlLysozyme. The soluble portion of each lysate (this will be referred toas the “lysate” from this point forward) was isolated by collectingsupernatant following centrifugation at full speed in a microcentrifuge.Lysates were then dialyzed into various buffers, whose compositionyielded the final reaction conditions indicated in figures upon dilutionof 1 μl of the dialyzed product with 8 μl dialysis buffer and 1microliter of probe diluted in water. Dialyzed lysates were either usedimmediately, or aliquoted and stored at −20° C.

Fluorescence Plate-Reader Assays

1 μl of a stock solution of 500 μM of the indicated probe was firstdiluted with 9 μls of high performance liquid chromatography (HPLC)grade water. One microliter dialyzed lysate (1 μg/μl) and 8 μl dialysisbuffer were added to 1 μl of the diluted probe. The reaction wasincubated at 37° C. for the time indicated. After incubation, 290 μl ofstop solution (10 mM EDTA+10 mM EGTA in DPBS without divalent cations)was then added to each reaction and fluorescence of reactions wasmeasured in triplicate (95 μls/well) in a fluorescence plate-reader(Analyst HT).

Bacterial Numbers Calculations

For the experiment shown, the OD600 of an overnight culture of thecoliform bacterial strain, diluted 1:10 was found to be 0.574. Using8×10⁸ cells/(ml*OD600) as a conversion factor, this amounts to10*0.574*8×10⁸=4.6×10⁹ cells/ml. 1 ml of this culture was pelleted bycentrifugation and the pellet was lysed in 1 ml buffer. The lysate wasdialyzed and then diluted 1:1,000, 1:3,000, 1:5,000, 1:7,000 and1:10,000 in dialysis buffer. One microliter of each dilution was usedper 10 μl reaction, each of which was divided into 3 wells of a 96 wellplate for fluorescence measurements. The number of bacterial cells perwell of the plate was calculated as in the following example (for the1:1,000 dilution): 1 μl*4.6×10⁹ cells/ml*(1 ml/1,000μl)*(1/1,000)*(1/3)=1,531 cells/well.

Zymograms

30 μg of each E. coli lysate were run on 12% acrylamide SDS-PAGE gelspolymerized with 1 nmol salmon sperm DNA (Invitrogen) per 8 ml gelmixture. Nucleases were activated by a series of washes. The first washcontains 12 mM MgCl₂ and 2.5% Triton X-100. The second wash contains 12mM MgCl₂, 2.5% TX-100, 100 mM NaCl, 1 mM DTT and 50 mM Tris-HCl pH 8.0.The third wash has 12 mM MgCl₂, 100 mM NaCl, 1 mM DTT and 50 mM Tris-HClpH 8.0. The gels were then incubated in the third wash for 2 hrs at 37°C. Gels were stained with SYBR Gold for 30 minutes and visualised with aUV-light transilluminator.

Bacterial Strains

Coliform ((Migula) Castellani and Chalmers, FDA strain Seattle 1946,ATCC #25922), wildtype (parental) E. coli (K-12 Catalog # NC0451794) andEndA (catalog #s NC0493574 and OEC4987200828144), nfi (catalog #OEC4987-200829641) and nth (catalog # OEC4987-213606177) KO strains(Thermo-Fisher).

Western Blotting

Western blotting was used to assess expression of Endonuclease 1 in theE. coli lysates. Lysates were resolved on 12% acrylamide SDS-PAGE gelsand transferred to a PVDF membrane. The membrane was blocked 2 hours in5% milk diluted in TBS+0.2% NP-40. The membrane was then incubatedovernight at 4° C. with a rabbit anti-Endonuclease 1 antibody (diluted1:5000 in milk), washed 3 times in TBS+0.2% NP-40, then incubated withgoat anti-rabbit HRP secondary antibody (1:5000 in TBS-T) 1 hour at roomtemperature. After washing 3× in TBS+0.2% NP-40, the membrane wasdeveloped with ECL.

Example 2

Urinary tract infections (UTIs) are thought to be the most common typeof bacterial infection and they are also the most common type ofhospital-acquired infection (Foxman, B. Epidemiology of urinary tractinfections: incidence, morbidity, and economic costs. Am J Med 113 Suppl1A, 5S-13S (2002); Wilson, M. L. & Gaido, L. Laboratory diagnosis ofurinary tract infections in adult patients. Clin Infect Dis 38,1150-1158 (2004)). The predominant pathogen responsible for theseinfections is E. coli (Foxman, B. The epidemiology of urinary tractinfection. Nat Rev Urol 7, 653-660 (2010); Kaper, J. B., Nataro, J. P. &Mobley, H. L. Pathogenic Escherichia coli. Nat Rev Microbiol 2, 123-140(2004)). If left untreated, UTIs of the lower urinary tract can progressto very serious and life-threatening conditions, including infections ofthe kidneys (pyelonephritis) and blood (bacteremia) (Kaper, J. B.,Nataro, J. P. & Mobley, H. L. Pathogenic Escherichia coli. Nat RevMicrobiol 2, 123-140 (2004).).

Upon initial clinical evaluation of suspected UTIs, rapid diagnosis andidentification of the causative bacterial species would enable the earlyadministration of an appropriate therapeutic reagent and thereby reducethe number of such serious infections. For instance, rapididentification of E. coli in clinical urine samples would enablephysicians to quickly select antibiotics based on established antibioticresistance profiles of E. coli strains found to cause UTIs in theregion. However, current clinical diagnostic methods do not providerapid identification of bacterial pathogens responsible for UTIs.Microbiological diagnostic methods require culture and take at least 24hours to reliably identify the responsible bacterial pathogen (Wilson,M. L. & Gaido, L. Laboratory diagnosis of urinary tract infections inadult patients. Clin Infect Dis 38, 1150-1158 (2004)). Urinalysismethods (e.g., measures of nitrite or leukocyte esterase) lack thedesired sensitivity and specificity for reliable diagnosis of UTIs anddo not identify the causative bacterial species (Wilson, M. L. & Gaido,L. Laboratory diagnosis of urinary tract infections in adult patients.Clin Infect Dis 38, 1150-1158 (2004)).

Detection of E. coli with the nuclease-activated probes of the presentinvention is sufficiently rapid (<3 hours) and sensitive to address thisunmet need for a novel clinical diagnostic assay for E. coli UTIs. Thediversity of nucleases found in nature also suggests that an assayspecific for E. coli (versus the nucleases present in other pathogensthat cause UTIs) allows for tailoring the makeup of the probe and thedigestion conditions to specifically allow Endonuclease I of E. coli toyield probe digestion.

Example 3

Food poisoning can be caused by eating food contaminated with bacteria,such as with Salmonella or E. coli. Food, such as beef, poultry, milk oreggs, may be contaminated during food processing or food handling. Thepresent method can easily detect contaminating bacteria on site, such asat a processing plant.

Example 4

The present invention provides, in certain embodiments, a means ofrapidly diagnosing Staphylococcus aureus bacteremia, a common medicalcondition with a very high mortality rate. (van Hal, S. J. et al.Predictors of mortality in Staphylococcus aureus Bacteremia. Clinicalmicrobiology reviews 25, 362-386, doi:10.1128/cmr.05022-11 (20012);Klevens, R. M. et al. Invasive methicillin-resistant Staphylococcusaureus infections in the United States. Jama 298, 1763-1771,doi:10.1001/jama.298.15.1763 (2007)). Bacteremia is a condition in whichviable bacteria are found in the blood circulation. Current diagnosticmethods for S. aureus bacteremia require time-consuming culturingmethods that take 24-48 hours. Rapid diagnosis of this condition wouldfacilitate the administration of effective antibiotic therapy at earliertimes and is expected to substantially reduce the mortality rate. Theutility of a quenched fluorescent, nuclease-activated oligonucleotideprobe-based approach for detecting bacterial pathogens via theirnuclease activities (Example 1 above; Hernandez, F. J. et al.Noninvasive imaging of Staphylococcus aureus infections with anuclease-activated probe. Nature medicine 20, 301-306,doi:10.1038/nm.3460 (2014). These probes are short oligonucleotides withdark quenchers coupled to their 3′-ends which suppress the fluorescenceof a fluorophore on the 5′-end when the probes are intact due to theclose proximity of quenchers to the fluorophore. Upon cleavage of theprobe by a nuclease, the quenchers diffuse away from the fluorophoresresulting in probe activation through unquenching of the fluorophore.These probes can be engineered through nucleotide modifications to beselectively digestible (and thus activatable) by target nucleases ofbacterial pathogens.

To explore whether probes engineered to specifically detect S. aureusvia the activity of its secreted nuclease (known as micrococcalnuclease) might enable the diagnosis of S. aureus bacteremia, the probeswere incubated with plasma of patients with confirmed S. aureusbacteremia and measured fluorescence. It was not possible to detect thenuclease activity in these specimens. This is perhaps not surprisingconsidering that bacteremia typically occurs with a very small number ofbacteria per unit volume of blood (i.e., <10 bacterial cells per 5 ml ofblood).

The sensitivity of this assay was determined by measuring the activityof various concentrations of purified micrococcal nuclease, diluted inbuffer. The assay sensitivity was also evaluated in the context of humanserum by preparing dilutions of purified micrococcal nuclease in humanserum and carrying out the assay with this material. Interestingly, itwas found that the assay was substantially less sensitive in human serumthan in buffer. This suggested that there are components of human serumthat inhibit the activity of the nuclease. Indeed, antibodies thatinhibit the catalytic activity of micrococcal nuclease were recentlyfound to be common components of human serum. (Schilcher, K. et al.Increased neutrophil extracellular trap-mediated Staphylococcus aureusclearance through inhibition of nuclease activity by clindamycin andimmunoglobulin. The Journal of infectious diseases 210, 473-482,doi:10.1093/infdis/jiu091 (2014)).

Micrococcal nuclease has been known for decades to be resistant toinactivation by heat denaturation when calcium and other proteins arepresent. (Cuatrecasas, P., Fuchs, S. & Anfinsen, C. B. Catalyticproperties and specificity of the extracellular nuclease ofStaphylococcus aureus. J Biol Chem 242, 1541-1547 (1967)). The inventorspostulated that heat treating human serum that contains micrococcalnuclease might result in inactivation of antibodies or other componentsthat inhibit micrococcal nuclease while leaving micrococcal nucleasefunctional. To evaluate this idea, micrococcal nuclease was seriallydiluted into human serum that was previously dialyzed into a buffercontaining 10 mM CaCl₂. Samples of each dilution were then either heattreated or untreated. Supernatants of centrifuged, heat treated serumwere then compared side-by-side with untreated serum in a nucleaseactivity assay with a quenched fluorescent oligonucleotide probe. Asshown in FIG. 12, this heat protocol unmasked micrococcal nucleaseactivity in the serum, with the effects being most evident at the lowerconcentrations of the nuclease.

The expectation that very low concentrations of the nuclease are presentin the blood of patients with S. aureus bacteremia provided a rationalefor the pursuit of further increases in assay sensitivity. One approachis to purify and concentrate the nuclease from serum specimens prior toincubation with the nuclease probes. Affinity-based approaches wereevaluated for nuclease concentration because these can be rapid and alsoprovide an additional degree of specificity for micrococcal nucleaseover non-target nucleases. A custom monoclonal antibody for micrococcalnuclease was produced (Pierce Biotechnologies). The supernatants ofseveral hybridoma clones were screened to identify those that producedantibodies that could effectively immunoprecipitate micrococcal nucleasewithout inhibiting its activity. Next, a purified rat monoclonalantibody with these desired properties was obtained. After using thisantibody with magnetic protein G-coupled beads to immunoprecipitatemicrococcal nuclease from dilute solutions, the nuclease-bound beadswere incubated directly (i.e., in suspension) with nuclease probes androbust probe activation was observed. This indicated not only that thenuclease was effectively immunoprecipitated, but that it retained itsactivity while bound to the antibody. The fact that the nuclease isfunctional when bound to the antibody allowed the elimination of anuclease elution step prior to probe digestion, thus providing for amore rapid assay.

The sensitivity of an assay for micrococcal nuclease was then evaluatedin which human plasma containing various amounts of the nuclease wasfirst heat treated and then subjected to immunoprecipitation, followedby probe incubation. The dialysis step used in the initial evaluation ofthe heating protocol was circumvented by spiking calcium chloridedirectly into the plasma prior to heating. With a one hour precipitationstep and a one hour probe incubation step, this assay took a total ofless than three hours. Immunoprecipitation of the nuclease after heatingprovided a robust improvement in assay sensitivity over heating alone,as shown in FIG. 13. In particular, samples that were only heat-treatedyielded background fluorescence levels (i.e., levels of the no nucleasecontrol samples) with micrococcal nuclease concentrations of 24.7femtomolar or less while heat-treated and immunoprecipitated samplesyielded levels above background levels with nuclease concentrations aslow as 247 attomolar.

Next, it was sought to demonstrate the efficacy of the assay as adiagnostic for S. aureus bacteremia. Heparinized plasma specimens wereobtained from two groups of individuals. The first group had S. aureusbacteremia, as confirmed by conventional blood culturing methods carriedout at the University of Iowa Hospital. Table 4 lists the time elapsedbetween the initiation of these blood cultures and detection ofbacterial growth in them; this time-to-positive value is considered arough indication of the bacterial load in the blood, with shorter timesindicative of larger bacterial loads.

TABLE 4 Specimen ID Time to Positive K Aerobic - 15 hr, 37 minAnaerobic - 15 hr, 37 min G Aerobic - 17 hr, 53 min J Anaerobic - 19 hr,15 min M Aerobic - 20 hr, 11 min P Aerobic - 20 hr, 15 min V Aerobic - 1day, 5 hr, 28 min B Aerobic - 1 day, 5 hr, 53 min D Aerobic - 1 day, 9hr, 26 min U Aerobic - 2 days, 10 hr, 25 min A Presumed Negative; NotTested F Presumed Negative; Not Tested H Presumed Negative; Not Tested IPresumed Negative; Not Tested L Presumed Negative; Not Tested N PresumedNegative; Not Tested R Presumed Negative; Not Tested S PresumedNegative; Not Tested T Presumed Negative; Not Tested Information on thespecimens used in FIG. 14. “Time-to-Positive” indicates the time elapsedfor the blood cultures of the same patients to indicate bacterialgrowth. Note: both aerobic and anaerobic cultures are prepared. In caseswhere only one of these became positive, only the positive value isincluded. “Presumed Negative” indicates that these specimens were drawnfrom individuals that were not exhibiting signs of active infections; noblood cultures were prepared from these individuals.

It is important to note that the blood used to prepare the plasma forthe assays was drawn on the same day as the blood drawn for thediagnostic blood cultures. The second group of individuals was selectedas control subjects based on the fact that they were not exhibiting anysigns of active infections. Because diagnostic culturing assays were notcarried out for these individuals, the plasma from them was classifiedas “presumed uninfected.”

A total of nine S. aureus positive (infected) and nine negative(presumed uninfected) human plasma specimens were tested in fivedistinct experiments. The data from these experiments is compiled inFIG. 14. Note that the fluorescence values of the plasma samples werenormalized by dividing by the values of “no plasma” control sampleswhich were included in each experiment to yield “activation ratios.”These ratios demonstrate a clear difference between the infected andpresumed uninfected specimens as all of the infected ratios are higherthan all of the presumed uninfected ratios. Collectively, the infectedplasma specimens have an average ratio that is seven-fold greater thanthat of the presumed uninfected specimens. In summary, a rapid (<3hours) nuclease activated probe-based assay has been developed that candetect the presence of S. aureus in clinical human plasma specimens ofbacteremic patients, and therefore forms the basis of a valuableclinical diagnostic assay for S. aureus bacteremia.

Methods

PolyTT Quenched Fluorescent Probe

PolyTT is an 11-mer DNA probe with a sequence of/6-FAM/TTTTTTTTTTT/ZEN/IAbRQSp/(SEQ ID NO: 5), where 6-FAM is afluorophore (fluorescein amidite), the T's indicate deoxythymidine (DNA)nucleotides, ZEN is the ZEN dark quencher, and IAbRQSp is the Iowa Blackdark quencher. The probe sequence is listed from the 5′- to the 3′-end.Its molecular weight is 4942.6 Da. The polyTT probe was synthesized andHPLC purified by Integrated DNA Technologies (IDT) of Coralville, IowaUpon receipt of the lyophilized probe from IDT, the probe was eitherstored directly at −80° C. or dissolved in TE (Ambion catalogue #:AM9849-10 mM Tris-HCl pH 8.0 and 1 mM EDTA) to a final concentration of500 μM, aliquoted into 1 μl volumes, and then stored at −80° C. Thisprobe serves as a substrate for micrococcal nuclease of S. aureus.

Evaluation of Heat Protocol in Human Serum

To evaluate the thermostability of micrococcal nuclease, non-targetnucleases and inhibitory antibodies in serum, human serum pooled fromhealthy donors was used (Bioreclamation IVT catalogue #: HMSRM, pooledhuman serum, no filtration). Serum specimens were dialyzed prior toexperiments as follows. 110 μl serum was dialyzed against 1.4 ml of 50mM Tris-HCl pH 9.0, 10 mM CaCl₂ (prepared from Sigma catalogue #:T2819—1 M Trizma hydrochloride solution, pH 9.0 and Sigma catalogue #:21115—1 M CaCl₂ solution) on a rocker at 4° C. for 2 hours with amicrodialysis tube (Pierce Biotechnology, Inc. catalogue #: 88262,96-well Microdialysis Plate 3.5K MWCO). The dialysis buffer wasexchanged for fresh buffer and the serum was then dialyzed for a second2 hour period. This dialysis protocol was replaced with alternativemethods in some experiments as described in Preparation of PlasmaSamples from S. aureus Bacteremic Patients and Control Subjects. Becausethe serum used here was not infected, it did not initially contain anymicrococcal nuclease. Defined amounts of a pure preparation ofmicrococcal nuclease were added to the dialyzed serum to evaluate thethermostability of the enzyme. Prior to addition to serum, the purenuclease was pre-diluted into 50 mM Tris-HCl pH 9.0, 10 mM CaCl₂ from a10 unit/pi stock solution (purified micrococcal nuclease was obtainedfrom Worthington catalogue #: LS004798—Nuclease, Micrococcal 45 ku;stock buffer consisted of 50% glycerol, 50% DPBS, prepared from Gibcocatalogue #: 14190-144—Dulbecco's phosphate-buffered saline, no calcium,no magnesium). 11.1 μl of each micrococcal nuclease dilution was addedto 100 μl of dialyzed serum in a 1.5 ml low protein binding microfugetube (Eppendorf catalogue #: 022431081—Protein LoBind Tube 1.5 ml). A“no nuclease” control sample was prepared with 11.1 μl of 50 mM Tris-HClpH 9.0, 10 mM CaCl₂ buffer and 100 μl of dialyzed serum. Samples weredivided in half, with one half to undergo the heating protocol and theother half to be reserved as unheated controls. The unheated controlsamples were placed at 4° C. during the heating protocol. The othersamples were then placed in a 90° C. heat block for 20 minutes. Theheated serum samples, which became cloudy upon heating due to proteinprecipitation, were then centrifuged at 17,000×g for 10 minutes. 1 μl ofpolyTT probe (synthesized and purified by IDT, see PolyTT QuenchedFluorescent Probe for description) diluted to a concentration of 50 μMin 50 mM Tris-HCl pH 9.0, 10 mM CaCl₂, was then added to 9 μl of eachheat-processed human serum supernatant and unheated control human serumsample. The tubes were incubated in the dark at 37° C. for 1 hour. Tostop the digestion by micrococcal nuclease, which requires calcium foractivity, 290 μl of 10 mM EDTA and 10 mM EGTA in DPBS (prepared fromAmbion catalogue #: AM9260G—0.5 M EDTA pH 8.0, Bio-World Catalogue #:40520008-1—0.5 M EGTA pH 8.0, and Gibco catalogue #:14190-144—Dulbecco's phosphate-buffered saline, no calcium, nomagnesium) was added to each tube. The stopped reactions were mixed bypipetting up and down, and then 90 μl was transferred to each of 3 wellsof a 96-well plate (Thermo Scientific catalogue #: 237105—Nunc F96MicroWell Black Polystyrene Plate) for triplicate readings. Fluorescencewas measured in a fluorescence plate-reader (Analyst HT or BiotekSynergy Mx) at 485/530 nm excitation/emission.

Preparation of Plasma Samples from S. aureus Bacteremic Patients andControl Subjects

Heparinized plasma specimens obtained from patients with confirmed S.aureus bacteremia or from individuals exhibiting no signs of activeinfections, were provided by the University of Iowa Tissue ProcurementCore Facility. These specimens were aliquoted into 110 μl volumes andstored at −80° C. For the assays, six aliquots of each specimen werethawed at 25° C. and combined into a single 660 μl sample in a 1.5 mllow protein binding microfuge tube. To enable micrococcal nucleasethermostability, 6.7 μl of 1 M CaCl₂ solution (Sigma catalogue #:21115—1 M CaCl₂ solution) was added to the plasma, yielding a finalconcentration of approximately 10 mM CaCl₂. Samples were then placed ina 90° C. heat block for 20 minutes. The plasma samples, which becamecloudy upon heating due to protein precipitation, were then centrifugedat 17,000×g for 10 minutes. Supernatants were transferred to fresh tubesand used for the subsequent immunoprecipitation/nuclease assay.

For comparison of assay sensitivity with and without theimmunoprecipitation step, 100 μl of various dilutions of micrococcalnuclease diluted in 50 mM Tris-HCl pH 9.0, 10 mM CaCl₂ and 11 μl of 1 MCaCl₂ were added to 1 ml of pooled human plasma (Bioreclamation IVTcatalogue #: HMPLLIHP, pooled human plasma, lithium heparinanticoagulant, no filtration). The samples were heated and centrifugedas described above and 500 ul of each supernatant was used forsubsequent immunoprecipitations or supernatants were used directly innuclease assays.

Immunoprecipitation of Micrococcal Nuclease from Plasma

Protein G-coupled magnetic beads (Life Technologies catalogue #:10004D—Dynabeads Protein G for Immunoprecipitation) were resuspended inthe manufacturer's vial by rotating the vial at room temperature for 5minutes. For each plasma sample, 40 μl of the beads suspension was addedto an empty 1.5 ml low protein binding microfuge tube. An additionaltube was prepared in the same way in parallel for use as a “no plasma”control. The tubes were placed on a magnet for ˜1 minute to separate thebeads from the manufacturer's storage solution, and the solution wasremoved with a pipette. The beads were resuspended in 1 ml of 0.02%Tween-20 in DPBS (wash buffer). This was prepared from Amresco catalogue#: 0777—Tween-20 Reagent Grade and Gibco catalogue #:14190-144—Dulbecco's phosphate-buffered saline, no calcium, nomagnesium. The tubes were then placed on the magnet to separate thebeads from the wash buffer, and the buffer was removed with a pipette.This washing step (re-suspending beads in wash buffer and removing washbuffer) was repeated once for a total of two washes. 50 μl ofanti-micrococcal nuclease monoclonal antibody (Pierce Biotechnology,Inc. custom produced antibody, stored in 50% glycerol at 0.75 mg/ml) wasdiluted with 450 μl wash buffer and used to resuspend the beads in eachbeads-containing tube. The tubes were incubated with beads and antibodyon a rotator at room temperature for 1 hour and then placed on themagnet to separate the beads from the antibody solution. The solutionwas removed with a pipette. The beads were then washed with wash buffertwice as described above. Next, each heat-processed human plasmasupernatant (prepared as described in Preparation of Plasma Samples fromS. aureus Bacteremic Patients and Control Subjects) was added to anantibody/beads-containing tube. The entire supernatant from each ofthese samples was used; the volume varied from approximately 300 to 350μl. 350 μl of 50 mM Tris-HCl pH 9.0, 10 mM CaCl₂ was added to thebeads-containing tube reserved for the “no plasma” control. For theside-by-side comparison of the assay sensitivity with and withoutimmunoprecipitation, 500 ul of each supernatant was used. The tubes wereincubated on a rotator at room temperature for 1 hour. Next, the tubeswere placed on a magnet to separate the beads from the heat-processedplasma supernatants or buffer solution, and the solutions were removedwith a pipette. The beads were then washed with 1 ml wash buffer (asdescribed above) a total of 3 times, using a fresh low protein bind tubefor each wash. The beads were then washed twice with 1 ml 50 mM Tris-HClpH 9.0, 10 mM CaCl₂, using a fresh low protein bind tube each time.

Fluorescence Plate-Reader Assay for Immunoprecipitated MicrococcalNuclease

Probe incubation reactions and plate-reader measurements ofimmunoprecipitated nuclease samples were carried out as follows. Eachbead sample (prepared as described in Immunoprecipitation of MicrococcalNuclease from Plasma) was resuspended in 60 μl 50 mM Tris-HCl pH 9.0, 10mM CaCl₂. 1 μl of polyTT probe (synthesized and purified by IDT, seePolyTT Quenched Fluorescent Probe for description) diluted to aconcentration of 50 μM in 50 mM Tris-HCl pH 9.0, 10 mM CaCl₂, was thenadded to each beads suspension. The tubes were incubated with probe on arotator at room temperature in the dark for 1 hour. The tubes were thenplaced on a magnet, separating beads from probe solution, and 50 μl ofeach probe-containing supernatant was transferred to a well of a 96-wellplate (Thermo Scientific catalogue #: 237105—Nunc F96 Micro Well BlackPolystyrene Plate). Fluorescence was measured in a fluorescenceplate-reader (Analyst HT or Biotek Synergy Mx) at 485/530 nmexcitation/emission.

Example 5 Degradation of Nuclease-Stabilized RNA Oligonucleotides inMycoplasma-Contaminated Cell Culture Media

Artificial RNA reagents such as siRNAs and aptamers often must bechemically modified for optimal effectiveness in environments thatinclude ribonucleases. Mycoplasmas are common bacterial contaminants ofmammalian cell cultures that are known to produce ribonucleases. Here,the inventors describe the rapid degradation of nuclease-stabilized RNAoligonucleotides in an HEK cell culture contaminated with Mycoplasmafermentans, a common species of Mycoplasma. RNA with 2′-fluoro- or2′-O-methyl-modified pyrimidines was readily degraded in conditionedmedia from this culture, but was stable in conditioned media fromuncontaminated HEK cells. RNA completely modified with 2′-O-methyls wasnot degraded in the Mycoplasma contaminated media. RNA zymogram analysisof conditioned culture media and material centrifuged from the mediarevealed several distinct protein bands (ranging from 30 to 68 kDa)capable of degrading RNA with 2′-fluoro- or 2′-O-methyl-modifiedpyrimidines. Finally, the Mycoplasma-associated nuclease was detected inmaterial centrifuged from the contaminated culture supernatants in aslittle as 15 minutes with an RNA oligo containing 2′-O-methyl-modifiedpyrimidines and labeled with a 5′-FAM and 3′-quencher. These resultssuggest that Mycoplasma contamination may be a critical confoundingvariable for cell culture experiments involving RNA-based reagents, withparticular relevance for applications involving naked RNA (e.g.,aptamer-siRNA chimeras).

Synthetic RNA that is exposed to cells or tissues must be protected fromribonuclease degradation in order to carry out its intended function inmost cases. Common approaches for avoiding nuclease degradation includenanoparticle encapsulation which insulates the RNA from exposure toribonucleases and chemical modification to render it resistant todegradation. Modification of RNA by substituting O-methyl or fluorogroups for the hydroxyl at the 2′-position of the ribose can greatlyenhance its stability in the presence of extracellular mammalianribonucleases.

These modifications are widely employed in the development of siRNAs andRNA aptamers for both research and therapeutic applications. siRNAs canbe modified with 2′-O-methyl substitutions in both sense and antisensestrands without loss of silencing potency, but only a subset ofnucleotides are typically modified with 2′-O-methyls asover-modification of the siRNA can reduce or eliminate its silencingability. siRNAs with 2′-fluoro modified pyrimidines have also beenreported to retain silencing activity in vitro as well as in vivo.

RNA with 2′-fluoro modified pyrimidines is the most commonly usedchemistry for development of RNA aptamers with potential therapeuticapplications. Such RNA is stable in animal serum and can also beefficiently transcribed in vitro with a mutant viral RNA polymerase,thus facilitating its use in the aptamer discovery process known asSELEX (Systematic Evolution of Ligands by EXpontential enrichment). Thestability of this RNA in other contexts such as conditioned cell culturemedia has not been well-studied.

Mycoplasmas are a genus of small bacteria that are common contaminantsof cell cultures. They lack a cell wall, are not susceptible to theantibiotics usually employed in cell culture and often go undetected incell culture due to their small size. Some Mycoplasma species, notablyMycoplasma pneumoniae, are human pathogens. Various Mycoplasma speciesare known to produce ribonucleases and deoxyribonucleases; however, theability of these nucleases to degrade chemically modified RNAformulations has not been explored prior to the present work.

The inventors evaluated the stability of RNA with 2′-fluoro modifiedpyrimidines in cell culture media conditioned by human embryonic kidney293 (HEK) cells and found the RNA to be substantially degraded afterfairly brief incubations. It was subsequently determined that the HEKcells were contaminated with Mycoplasma, and it was investigated whetherthis contamination was responsible for the observed nuclease activity.

Materials and Methods

Cell Culture and Conditioned Media. HEK cells were inadvertentlycontaminated with Mycoplasma at some point during routine culturemaintenance. The contamination was later detected and confirmed by PCRto be Mycoplasma fermentans. This Mycoplasma contaminated cell line wasused as a positive control for Mycoplasma testing methods.Uncontaminated HEK cells were obtained from ATCC (ATCC-CRL-1573™).Contaminated and uncontaminated HEK cells were grown in DMEM (GIBCO)containing 10% heat-inactivated bovine serum, 50 U/ml penicillin, and 50μg/ml streptomycin at 37° C., 5% CO₂ in a moist atmosphere. Forpreparation of conditioned media, contaminated or uncontaminated HEKcells were grown to −80% confluency on 100 mm or 150 mm culture dishes.The culture media was then replaced with fresh media. After 48 hours ofincubation, the media was centrifuged for 6 minutes at 1,250 rpm in atable-top centrifuge to remove cellular debris. Finally, the supernatantwas transferred into a fresh tube and used as “conditioned media.”Unconditioned media had the same composition (see above), but was notincubated with cells. Particulate matter was centrifuged from suchconditioned media for the experiments described in FIG. 15C. 6milliliters of conditioned media was centrifuged at 13,300 rpm in amicrocentrifuge. The pellet was washed with PBS and then dissolved in 20μl 1% Triton X-100 in PBS. Eppendorf tubes with these reactions wereimaged with a digital camera and UV-light trans-illumination.

Mycoplasma Culture.

Mycoplasma broth consisted of 10% yeast extract solution (Gibco), 20%heat-inactivated fetal bovine serum, 70% heart infusion broth (BDBiosciences), 50 U/ml penicillin, and 50 μg/ml streptomycin. Afreeze-dried culture of Mycoplasma fermentans (ATCC #15474) wasrehydrated in 10 ml of Mycoplasma broth. Several 10-fold serialdilutions of this culture were then prepared and the bacteria were grownin 50 ml conical tubes at 37° C. for several days. For the experimentshown in FIG. 16, 1 ml of Mycoplasma fermentans culture grown for 5 dayswas pelleted at 13, 3000 rpms for 5 minutes. Supernatant was discardedand the lysate was prepared by dissolving the pellet in 20 μl 1% TritonX-100 in PBS. The lysate was incubated with RNAse substrates for 1 hourat 37° C.

Chemically Modified RNA.

The following 51 nucleotide long RNA sequence was used for the gel-baseddegradation assays and the RNA zymograms:5′-GGGAGGACGAUGCGGGACUAGCGAUCUGUUACGCACAGACGACUCGCCCGA-3′ (SEQ ID NO:34). Several versions of this RNA, with modifications as described infigure legends and the results section were used. FAM (fluoresceinamidite)-labeled versions were used in the gel-based degradation assays,whereas non-fluorescent versions were used for the zymograms. These RNAswere obtained from Trilink Biotechnologies (San Diego, Calif.).

Gel-Based Degradation Assay.

For each degradation assay sample, 6 μl of oligo (2 μM) was combinedwith 6 μl of unconditioned and conditioned media and incubated for 0.5,1, 2, or 4 hours at 37° C. After incubation, samples were combined with12 μl of loading buffer (formamide with 0.5×TBE), incubated for 6minutes at 65° C., transferred to ice for 5 minutes, briefly centrifugedand kept on ice until loading. Samples were run on a 7.7 M Urea/10%acrylamide gel at 100 volts for 80 minutes. Gel images were acquiredwith a Gel Doc™ XR+ System (Bio-Rad) with ultraviolet lighttransillumination and a standard fluorescence filter for imagingethidium bromide.

RNAse Substrate Plate-Reader Assays.

The RNAse substrates were synthesized by Integrated DNA Technologies(IDT; Coralville, Iowa). These probes consist of a 12 nucleotide longRNA oligo, 5′-UCUCGUACGUUC-3′ (SEQ ID NO: 6), with the chemicalmodifications indicated in figure legends, flanked by a FAM(5′-modification) and a pair of fluorescence quenchers, “ZEN” and “IowaBlack” (3′-modifications). For the RNA degradation assays, 1 μl of eachRNAse substrate (50 picomoles) was combined with 9 μl of sample (e.g.,conditioned media) and incubated at 37° C. for time points indicated inthe figures. After the incubation period, 290 μl of PBS supplementedwith 10 mM EDTA and 10 mM EGTA was added to each sample and 95 μl ofeach sample was loaded in triplicate into a 96-well plate (96Fnon-treated black microwell plate (NUNC)). Fluorescence intensity wasmeasured with a fluorescence microplate reader (Analyst HT; Biosystems).For the Triton X-100 lysate samples, the undiluted 10 μl reactions wereimaged in eppendorf tubes with a Gel Doc™ XR+ System (Bio-Rad) withultraviolet light transillumination and a standard fluorescence filterfor imaging ethidium bromide.

PCR.

All cell cultures were tested for Mycoplasma infection by PCR. Fourprimer sets, previously described (Choppa, P. C., Vojdani, A., Tagle,C., Andrin, R., and Magtoto, L. (1998). Multiplex PCR for the detectionof Mycoplasma fermentans, M. hominis and M. penetrans in cell culturesand blood samples of patients with chronic fatigue syndrome. Mol CellProbes 12, 301-308), were used to identify a conserved region among allmembers of the genus Mycoplasma and three specific species: Mycoplasmafermentans, Mycoplasma hominis and Mycoplasma penetrans. Mycoplasma,centrifuged from conditioned cell culture media, provided the templateDNA for the Mycoplasma specific PCRs. The Mycoplasma was isolated fromthe culture media as follows: conditioned media was centrifuged for 6minutes at 1,250 rpm to pellet cellular debris. 2 ml of the supernatantfrom this spin were then centrifuged for 5 minutes at 17,000×g to pelletany Mycoplasma present in the media. The pellet was re-suspended in 50μl of water; this served as the PCR template. The PCR reaction mixtureswere prepared in a total volume of 100 μl containing 10 μl of PCRtemplate, 2 μl dNTP's (10 μM), 1 μl of each primer (100 μM), 50 μl ofChoice Taq Mastermix (Denville Scientific) and 36 μl of water. 30 cyclesof PCR were carried out. The temperature steps were as follows: 94° C.for 5 minutes, 30× (denaturation at 94° C. for 30 seconds; annealing at55° C. for 30 seconds; elongation at 72° C. for 30 seconds) a finalextension step of 72° C. for 10 minutes was carried out at thecompletion of the cycling. The PCR products were analyzed on a 2% (w/v)agarose gel stained with 0.5 μg/ml ethidium bromide. The DNA bands werevisualized with a Gel Doc™ XR+ System (Bio-Rad) with ultraviolet lighttransillumination and a standard fluorescence filter for imagingethidium bromide.

DAPI Staining.

Mycoplasma was visualized in cultured cells via DAPI(4′,6-diamidino-2-phenylindole, dihydrochloride, Invitrogen) staining.Briefly, HEK cells were grown on glass bottomed culture dishes fromMatTek (Ashland, Mass.). Cells were then fixed by incubation for 20minutes at −20° C. in 100% methanol and stained with DAPI following theprotocol provided in the DAPI product insert. The cells were then rinsedseveral times in PBS and fluorescence was visualized with an OlympusIX71 fluorescence microscope equipped with a 40× oil-immersionobjective, fluorescence filters appropriate for DAPI and a cooled CCDdigital camera.

DNA Zymograms.

Mycoplasma-free and Mycoplasma-positive cell cultures were serum-starvedfor 48 hours on 150 mm culture dishes in 20 ml of DMEM. Media wascollected, spun at 1,250 rpms for 6 minutes to eliminate cell debris,then spun at 17,000×g for 5 minutes to pellet Mycoplasma from theconditioned media. These pellets were solubilized in 50 μl SDS sampleloading buffer. The supernatants from the second centrifugation wereconcentrated with a YM-10 Amicon centrifugal filter device (MW cutoff of10 kDa) to a final volume of approximately 100 μl. One or 3 μl of theconcentrated media or pellet, respectively, were loaded per lane of an8% acrylamide SDS gel containing 200 μg/ml salmon sperm DNA(Invitrogen). After the gel was run, nucleases were activated by aseries of 10 minute washes: 2 washes in 2 mM CaCl₂, 2 mM MgCl₂, 2.5%Triton X-100 in water; 2 washes in 2 mM CaCl₂, 2 mM MgCl₂, 2.5% TritonX-100 in 50 mM Tris-HCl (pH 7.4); 2 washes in 2 mM CaCl₂, 2 mM MgCl₂ in50 mM Tris-HCl (pH 7.4). The gels were then incubated in 2 mM CaCl₂, 2mM MgCl₂ in 50 mM Tris-HCl (pH 7.4) for either 2 hours or overnight at37° C. The gels were stained with 0.5 μg/ml ethidium bromide andvisualized with a Gel Doc™ XR+ System (Bio-Rad) with ultraviolet lighttransillumination and a standard fluorescence filter for imagingethidium bromide.

Chemically Modified RNA Zymograms.

Samples were prepared as for the DNA zymograms (described above).Proteins were separated on 8% acrylamide SDS gels polymerized witheither 550 nM of an RNA oligo with 2′-fluoro-modified pyrimidines or 250nM of an RNA oligo with 2′-O-methyl-modified pyrimidines. See“Chemically Modified RNA” above for the sequence of these oligos. Gelswere washed as above, but stained with a 1:10,000 dilution of Sybr Goldnucleic acid gel stain (Invitrogen) and visualized with a Gel Doc™ XR+System (Bio-Rad) with ultraviolet light transillumination and a standardfluorescence filter for imaging ethidium bromide.

Results

To evaluate the stability of RNA with 2′-fluoro modified pyrimidines inconditioned culture media, a 51 nucleotide RNA with 2′-fluoro modifiedpyrimidines (underlined) and a 3′-FAM(5′-GGGAGGACGAUGCGGGACUAGCGAUCUGUUACGCACAGACGACUCGCCCGA-3′-FAM (SEQ IDNO:35)) was incubated in serum-containing media, conditioned with an HEKcell culture recently obtained from ATCC, or with an older HEK cellculture. As previously reported, this RNA was found to be resistant tonuclease degradation in the presence of animal serum. While the modifiedRNA was also stable in conditioned media from the HEK cells obtainedfrom ATCC, the conditioned media from the older HEK cells almostcompletely degraded it after a 4-hour incubation at 37° C. The RNA wasthen resolved on a urea/acrylamide denaturing gel and imaged with adigital camera and UV-light trans-illumination; the oligo was labeled onits 3′-end with FAM. PCR primer sets specific for genomic components ofMycoplasma fermentans, Mycoplasma hominis, Mycoplasma penetrans, or fora region of the Mycoplasma genome that is conserved within the genus,were used to amplify DNA present in conditioned culture media. ExpectedPCR product sizes are as follows: Mycoplasma genus PCR: 280 bp;Mycoplasma fermentans: 206 bp; Mycoplasma hominis: 170 bp; Mycoplasmapenetrans: 470 bp. The PCR assay for the presence of Mycoplasma detecteda DNA sequence conserved within the genome of the Mycoplasma genus inthe culture supernatant of the older HEK cell culture supernatant, butnot in the media from the more recently obtained culture. A DNA sequencespecific to the Mycoplasma fermentans species was also detected in mediafrom the older HEK cell culture, as indicated with a PCR product ofexpected size. Sequencing of this PCR product confirmed that theamplified sequence is derived from the Mycoplasma fermentans genome. Incontrast, neither Mycoplasma hominis nor Mycoplasma penetrans wasdetected.

As an additional means of detecting Mycoplasma, DAPI staining of HEKcells from these two cultures was carried out. Small, punctate,extra-nuclear DAPI-labeling, indicative of Mycoplasma contamination, wasseen throughout the older HEK culture, but only nuclear labeling wasseen in the culture obtained from ATCC. Together, these data demonstratethe presence of a ribonuclease that readily degrades RNA with 2′-fluoromodified pyrimidines in a Mycoplasma-contaminated but not in aMycoplasma-free HEK cell culture.

Next, the activity of this ribonuclease after heat pre-treatment wasstudied, in the presence of EDTA (a chelator of divalent cations) and inthe presence of a broad-spectrum ribonuclease inhibitor. The same RNAoligo initially examined was used for this experiment (51-mer with2′-fluoro-modified pyrimidines and a 3′-FAM) with conditioned media fromthe Mycoplasma-contaminated HEK cells. Results of this experimentindicate that the ribonuclease activity is sensitive to heat treatmentand is thus likely protein in nature. Like many ribonucleases, itsactivity is dependent on divalent cations as chelation of divalentcations with EDTA inhibited degradation of the modified RNA. Todetermine the dependence of the nuclease activity on divalent cations,conditioned media was incubated with RNA in the presence of 10 mM EDTA.A broad-spectrum RNAse inhibitor, Superase.in, was co-incubated (at 1unit/μl) with the RNA in conditioned media to determine the sensitivityof the nuclease activity to this reagent. The RNA was resolved on aurea/acrylamide denaturing gel and imaged with a digital camera andUV-light trans-illumination; the oligo is labeled on its 3′-end withFAM. The broad-spectrum RNAse inhibitor, Superase.in, did not have anapparent impact on the activity.

While the 2′-fluoro nucleotide modification is widely used for thedevelopment of RNA aptamer-based therapeutic approaches, othermodifications are more commonly used to protect synthetic RNAs in otherapplications such as RNAi. The 2′-O-methyl modification is widelyemployed and we thus examined the susceptibility of RNA with2′-O-methyl-modified nucleotides to degradation by theMycoplasma-associated ribonuclease activity. For this experiment, 3 RNAoligos were used. Each was 51 nucleotides in length, of identicalsequence and with a 3′-FAM. The first oligo had 2′-fluoro-modifiedpyrimidines (purines were unmodified) (described above), the second had2′-O-methyl-modified pyrimidines (purines were unmodified) and everynucleotide of the third oligo was modified with 2′-O-methyls.Co-incubation of these oligos with media conditioned by theMycoplasma-contaminated HEK cells for 30 minutes, 1 hour, 2 hours or 4hours again demonstrated the near-complete degradation of the oligo with2′-fluoro-modified pyrimidines. The oligo with 2′-O-methyl-modifiedpyrimidines was more resistant to degradation, but was almost completelydegraded after 4 hours. Finally, there was no detectable degradation ofthe oligo that was completely modified with 2′-O-methyls at any of thetime-points.

Additional cell lines, contaminated with distinct Mycoplasma species(i.e., non-Mycoplasma fermentans species) were also tested for nucleaseactivity against RNA oligos with 2′-O-methyl- and 2′-fluoro-modifiedpyrimidines. Some of these cell lines possessed strong nuclease activityin their supernatants while others did not.

To further characterize the Mycoplasma-associated ribonuclease,zymograms were carried out with unmodified DNA or RNA with 2′-fluoro- or2′-O-methyl-modified pyrimidines. For these experiments, serum-freeconditioned cell culture media was used that was concentrated withfilter centrifugation as well as detergent-lysed particulate mattercentrifuged from the conditioned media. Concentrated media or materialpelleted by centrifugation from media conditioned by Mycoplasma-free orMycoplasma contaminated HEK cells was resolved on 8% acrylamide SDS gelsembedded with DNA, RNA with 2% fluoro-modified pyrimidines or RNA with2′-O-methyl-modified pyrimidines. After running, the gels were washedwith SDS-free buffer containing divalent cations and incubated at 37° C.for 2 hours to allow nuclease digestion. The gels were stained withethidium bromide (DNA zymogram) or SYBR Gold nucleic acid gel stain andimaged with a digital camera and UV-light trans-illumination, revealingprotein bands with nuclease activity. The particulate matter presumablycontains Mycoplasma in the contaminated culture sample and was alsofound to possess ribonuclease activity.

Multiple protein bands present in the Mycoplasma-contaminated, but notthe Mycoplasma-free concentrated culture supernatants could be seen inall 3 zymograms. A cluster of bands that migrated between the 37 and 50kDa molecular weight markers was prominent in these samples. A smallerprotein, of approximately 30 kDa digested the modified RNAs; nodigestion was seen in this region of the DNA zymogram. A larger protein,of approximately 68 kDa, produced a band in the DNA zymogram, but not inthe modified RNA zymograms. However, longer digestion periods (e.g.,overnight) did yield a band of approximately this size in the modifiedRNA zymograms. The prominent cluster of bands between 37 kDa and 50 kDapresent in all of the zymograms suggests the presence of multiplenucleases with broad substrate specificities.

The detergent-lysed particulate matter produced a very different patternof bands on the zymograms. As with the concentrated supernatant, darkbands indicating digestion were only seen in the sample prepared fromthe Mycoplasma-contaminated culture. However, the patterns of bandsclearly indicate that there are multiple nucleases present in theparticulate matter of the culture media with distinct substratespecificities. The prominent cluster of bands between the 37 kDa and 50kDa molecular weight markers that was present in the concentratedculture supernatant was not seen in the particulate matter samples.

Because the Mycoplasma-associated nuclease can be distinguished fromendogenous mammalian nucleases by its distinct substrate specificity, wereasoned that the presence of mycoplasmas, in various contexts, might beinferred by the susceptibility of chemically modified ribonucleasesubstrates to degradation. While gel-based assays provide a simple andstraightforward means of detecting nuclease activity, a more rapid andsensitive assay for ribonucleases that degrade unmodified RNA has beendescribed. The basis for this assay is a short oligonucleotide RNAsesubstrate, end-labeled with a fluorophore on one end that is renderednon-fluorescent by its close proximity to a quencher on the other end(FIG. 15A) (Kelemen, B. R., et al. (1999). Hypersensitive substrate forribonucleases. Nucleic Acids Res 27, 3696-3701). Upon cleavage of thesubstrate, the quencher diffuses away from the fluorophore, which thenexhibits fluorescence.

This approach was adapted to detect the Mycoplasma-associated nucleaseby generating chemically modified RNAse substrates with fluorophore andquencher conjugates. Four different RNA chemistries were tested:2′-fluoro-modified pyrimidines, 2′-O-methyl-modified pyrimidines,complete 2′-fluoro modifications, and complete 2′-O-methylmodifications. Initially, each of these RNAse substrates was incubatedfor 4 hours with culture media conditioned by either Mycoplasma-free orMycoplasma-contaminated HEK cells (FIG. 15B). While the complete2′-O-methyl-modified substrate was not digested in either media, theother 3 RNAse substrates exhibited substantially greater fluorescenceafter incubation in the Mycoplasma-contaminated media. Of these, thesubstrate with 2′-O-methyl-modified pyrimidines exhibited the greatestrelative fluorescence increase between uncontaminated and contaminatedmedia. This substrate was thus characterized further.

Centrifugation of particulate matter from the Mycoplasma-contaminatedculture supernatants provided a simple and rapid means of obtainingconcentrated nuclease activity. The possibility that this approach mightincrease the sensitivity of Mycoplasma detection with RNAse substrateswas explored. The RNAse substrate with 2′-O-methyl-modified pyrimidineswas incubated with a detergent lysate of centrifuged particulate matterfrom Mycoplasma-free or Mycoplasma-contaminated HEK cells for 1 hour(FIG. 15C). Conditioned media from these cultures was compared inparallel. While both the lysate and conditioned media exhibited strongnuclease activity, the lysate produced a greater signal in this assay. Aclear signal could be seen over the background in this assay, even withan abbreviated (15 minutes) incubation (fluorescence was measured on anultraviolet light box in this case).

The contaminated HEK cell culture is a complex preparation as itcontains cells derived from two distinct organisms. While theuncontaminated HEK cells lack nuclease activity capable of efficientlydegrading RNA with 2′-fluoro-modified or 2′-O-methyl-modifiedpyrimidines, we also sought to measure such activity in a pure cultureof Mycoplasma fermentans. Consistent with the current observations ofthe contaminated HEK cell culture, lysates prepared from Mycoplasmafermentans bacteria exhibited robust nuclease activity against RNAsubstrates with 2′-fluoro-modified and 2′-O-methyl modified pyrimidines(FIG. 16). Nuclease activity against RNA substrates with2′-fluoro-modified and 2′-O-methyl-modified pyrimidines was also foundin the bacterial culture supernatant (not shown).

Discussion

It was found that conditioned media from HEK cells contaminated withMycoplasma fermentans possesses ribonuclease activity that readilydegrades RNA with 2′-fluoro-modified pyrimidines and2′-O-methyl-modified pyrimidines. Comparable ribonuclease activity wasseen in a pure culture of Mycoplasma fermentans, but not in anuncontaminated, HEK cell culture. These observations are consistent withthe conclusion that the activity in the contaminated HEK cell culture isderived from the Mycoplasma bacteria. Zymograms with chemically modifiedRNA revealed the presence of multiple protein bands (from ˜30 kDa to ˜68kDa) in the conditioned, Mycoplasma-contaminated culture media thatpossess this ribonuclease activity. Some of these proteins were found inparticulate matter in the media, presumably containing free-floatingMycoplasma. RNAse substrates synthesized with chemically modified RNAdetected the presence of this ribonuclease activity after a 15 minuteincubation.

This work was undertaken to better understand the stability ofchemically modified RNA oligonucleotides in cell culture settings. Theresults identify a critical variable for the use of RNA-based reagentsin cell culture experiments. While the potential for Mycoplasma-basedartifacts in cell culture experiments is widely known, many researchersdo not regularly test their cell lines for mycoplasmas. Lack of regulartesting is perhaps the primary reason that mycoplasmas continue to beproblematic for cell culture studies. It is estimated that 15-35% ofcell lines used are contaminated with mycoplasmas, with MycoplasmaFermentans among the most prevalent species identified in cell lines.

The manner in which Mycoplasma contamination affects experimentaloutcome varies depending on the nature of the experiment. The presentresults suggest that experiments involving the application of naked RNAdirectly to cells are particularly vulnerable to Mycoplasma-dependentRNA degradation and experimental failure. These experiments include thestudy or application of siRNAs, RNA aptamers and ribozymes. Forinstance, the delivery of siRNAs by directly coupling them to targetingreagents such as antibodies, aptamers, peptides and other syntheticligands all entail the application of naked RNA to cells. The evaluationof RNA aptamers targeting cell surface receptors or extracellulartargets such as growth factors in cell culture, likewise, involves theapplication of naked RNA to cells.

The characterization of the Mycoplasma-associated ribonuclease activitywith nucleic acid zymograms revealed the presence of multiple proteinswith deoxyribonuclease and ribonuclease activity in the contaminatedcell culture media. The presence of multiple bands in similar patternsin all 3 of the zymograms between the 37 kDa and 50 kDa molecular weightmarkers suggests that several nucleases capable of digesting DNA, or RNAwith 2′-fluoro- or 2′-O-methyl-modified pyrimidines are produced by theMycoplasma. Other bands found in the Mycoplasma-contaminated samplesexhibited substrate specific degradation among the 3 nucleic acidchemistries tested. Altogether, the results from the zymograms suggestthere are multiple Mycoplasma-derived ribonucleases present in thecontaminated culture media that can readily degrade RNA with either2′-fluoro- or 2′-O-methyl-modified pyrimidines. The identity of theseproteins has not been determined. Because zymograms depend on proteinrefolding following SDS-denaturation, it is possible that someribonucleases present in the culture media did not yield a signal on thezymograms.

The fraction of Mycoplasma species that produce ribonucleases capable ofdegrading chemically modified RNAs is uncertain. The fact that RNAoligos with 2′-fluoro- or 2′-O-methyl-modified pyrimidines were degradedin cultures contaminated with distinct, yet unidentified species ofMycoplasma indicates that the activity is not limited to the Mycoplasmafermentans species. Considering the diverse nature of mycoplasmas, it isperhaps not surprising that Mycoplasma-contaminated cell cultures thatlacked such robust nuclease activity were found.

Because many mycoplasmas are human pathogens, the detection ofMycoplasma-derived nucleases has clinical diagnostic applications. DNAseactivity has in fact been used to differentiate among various bacterialpathogens, including the identification of coagulase-positivestaphylococci, such as Staphylococcus aureus in bovine milk samples.Such assays depend on the isolation of the bacteria from biologicalfluids and tissues that also contain DNAses, which would otherwisegenerate background; isolation and culture of the bacteria can consumevaluable time. The use of chemically modified nucleic acids provides analternative that could be used in more clinically relevant settingswithout generating background. Indeed, a chemically modified RNAsesubstrate that rapidly and robustly detected Mycoplasma-derivedribonuclease activity in serum-containing conditioned media wasdeveloped; digestion of this RNAse substrate in the same mediaconditioned by an uncontaminated culture was minimal. Chemicallymodified nucleic acids can thus facilitate the rapid determination ofthe presence of bacterial contamination.

Example 6 In Vivo Detection

The cleavage of oligonucleotides can be visualized in various ways, butas discussed above, the inventors favor flanking the sequences with afluorophore and a quencher for rapid detection of the cleavage activitywith a fluorometer. Advancing one step further, the inventors envisioninjecting the fluorescent probe into patients and using fluorescentdetection to localize sights of infection (in addition to specificpathogen data) clearly within the patient. Preliminary data of this typehas been established in mouse models. Briefly, mice were injected withmicrococcal nuclease (purified Staph. aureus nuclease) in leg andinjected with probe in tail vein. This procedure resulted influorescence being seen at site of nuclease and subsequently in liver.

Example 7 In Vitro Detection

Nuclease Probe Plate-Reader Assay: The nuclease probes were synthesizedby Integrated DNA Technologies (IDT; Coralville, Iowa). These probesconsist of a 12 nucleotide long RNA oligo, 5′-UCUCGUACGUUC-3′ (SEQ IDNO:6), with the chemical modifications, flanked by a FAM(5′-modification) and a pair of fluorescence quenchers, “ZEN” and “IowaBlack” (3′-modifications. Three samples were assayed for degradation.PBS, 1 μl of each probe (50 picomoles) was combined with 9 μl of PBS.RNase A, 1 μl of each probe was combined with 8 μl of PBS and 1 μl RNaseA (˜50 U/μl). Micrococcal Nuclease (MN), 1 μl of each probe was combinedwith 8 μl of PBS and 1 μl of MN (10 U/μl). All the samples wereincubated at 37° C. for 4 hours. After the incubation period, 290 μl ofPBS supplemented with 10 mM EDTA and 10 mM EGTA was added to each sampleand 95 μl of each sample was loaded in triplicate into a 96-well plate(96F non-treated black microwell plate (NUNC)). Fluorescence levels areshown in FIG. 16 (these are the PMT (photomultiplier tube) values).Fluorescence was measured with a fluorometer.

A nuclease from a third pathogenic bacterium, Streptococcus pneumoniae,has also been evaluated. A nuclease is expressed on the membrane of theStreptococcus pneumoniae bacterium, making it easier to detect thannucleases that are secreted because it cannot diffuse away from thecell. The investigators found that this nuclease, which is known as EndAis capable of digesting a probe that has 2′-fluoro modified pyrimidines,but not a probe with 2′-O-methyl modified pyrimidines.

Degradation Activity of Micrococcal Nuclease and EndA Nuclease.

Unmodified (RNA and DNA) and modified (2′-Fluoro pyrimidines and2′-O-Methyl pyrimidines) nucleic acid substrates were used to assay thenuclease activity profile of Micrococcal Nuclease (MN) and EndA (H160G)Nuclease. The substrates were flanked by a fluorescent dye (FAM) at the5′-end and a quencher at the 3′-end. This approach allows the evaluationof nuclease activity which is indicated by increases in fluorescenceupon substrate digestion. 50 pmoles of substrate were incubated with MN(1 U/μL) and EndA H160G Nuclease (2 μM) in 10 μl total volume. Imadazolewas included in the EndA H160G reactions to recapitulate the enzymaticproperties of the wildtype enzyme. This mutant version of the enzyme wasused because the wt enzyme was toxic to E. coli and could not beproduced recombinantly in large amounts. 50 pmoles of each substrate andbuffer were used as controls. All reactions were incubated for 30minutes at 37° C. After incubation, 290 μl of buffer supplemented with10 mM EDTA and 10 mM EGTA were added to each sample and 95 μl of eachsample were loaded in triplicate into a 96-well plate (96F non-treatedblack microwell plate (NUNC)). Fluorescence intensity was measured witha fluorescence microplate reader (Analyst HT; Biosystems) (FIG. 17).

Example 8 Nuclease-Activated Probes for Imaging Staphylococcus aureusInfections

S. aureus infections are a major clinical problem that results in avariety of life-threatening and debilitating medical conditionsincluding septic joints, osteomyelitis and endocarditis. Development ofantibiotic resistant strains of S. aureus has compounded the difficultyof treating infections and highlights the need for novel antibiotics andbetter diagnostic approaches for their evaluation. Most S. aureusinfections with clinical significance are localized to internal tissuesor organs that are difficult to access. Definitive diagnosis of S.aureus infection thus necessitates testing biopsies of suspected tissuesfor presence of the bacteria. Because only limited tissues can besurveyed, biopsies offer only a limited assessment of the possibility anindividual is suffering from an S. aureus infection. In some cases, suchas endocarditis, biopsies are impractical and diagnoses are made withcircumstantial evidence such as heart murmur and the detection ofbacteria in the circulation.

To address the present shortcomings in the diagnostic technology forlocalized S. aureus (and other bacterial) infections, molecular imagingapproaches have been developed to non-invasively detect bacteria inanimals. These approaches depend on the selective affinity of theimaging reagents for components of bacteria. For example, one approachuses molecular probes that function by binding components of bacteriawith greater affinity than mammalian cells and tissues. In general,because such probes are always “on” they have suboptimaltarget-to-background ratios, which limit their sensitivity. These probesare limited by the fact that they produce signal prior to encounteringtheir target (i.e., they are not activatable probes) and most of themare non-specific with respect to bacterial species.

An alternative molecular imaging approach, involving quenchedfluorescent probes that are activated by tumor-specific proteases, hasprovided a valuable imaging platform for cancer imaging. Because suchactivatable probes do not produce signal (fluorescence) until the probeencounters its target, the result is a very high target-to-backgroundratio and a much more sensitive means of target detection. While thisapproach has proven valuable for imaging cancer, to-date it has not beenapplied to imaging bacteria, possibly due to a scarcity of appropriatebacteria-derived proteases. The inventors exploited the interfacebetween chemically modified nucleic acids and bacterial nucleases todevelop activatable imaging probes for bacterial infections. Thisresearch is innovative because the exploitation of nucleases for imagingis a novel direction, both for the imaging of bacterial infections inparticular and for whole-animal imaging in general.

The present invention provides a robust activated imaging probe-basedapproach for the non-invasive detection and localization of S. aureusinfections in animals. This contribution is significant becauseactivated imaging probes have critical advantages (e.g., hightarget-to-background ratios) over existing technology for this problem,and may prove to be generally useful for both research and clinicaldiagnostic applications involving S. aureus. The present approachfacilitates the evaluation in experimental animals of novel antibioticsfor naturally occurring strains of these bacteria. This near-infraredfluorescence-based imaging approach also is useful for the diagnosis andtreatment evaluation of S. aureus infections in humans. Indeed,near-infrared fluorescent dyes are currently used as clinical imagingtools for retinal angiography, cardiac function, hepatic output,sentinel lymph node dissection and colon polyp identification. Theclinical applicability of near-infrared imaging, which has limitedimaging depth in tissues (estimated at 7-14 cm), is expanding due to thedevelopment of medical imaging devices such as endoscopes withfluorescence imaging capabilities. Interestingly, many additionalproblematic pathogens are also known to express secreted or cell-surfacenucleases (e.g., Streptococcus pneumoniae), which are used for theirdetection.

A. Generation of Nuclease-Activated Probes that Specifically DetectMicrococcal Nuclease (MN) of S. aureus

The need for clinical diagnostic imaging of bacterial infections isgreatest for localized infections, which are often difficult todiagnose. In contrast, systemic infections can usually be detected withsimple blood tests. S. aureus is the most common bacterial cause of avariety of focal infections in humans, including infectious joints,osteomyelitis and endocarditis. For example, S. aureus is the causativebacterial pathogen in approximately half of the cases of infectiousjoints which are a serious medical condition, associated withsubstantial morbidity and mortality. Additional types of bacteria thatare commonly found to cause septic joints include non-aureusStaphylococci and Streptococci (group A-G and pneumoniae). A variety ofadditional bacterial species, including Gram negative bacteria such asE. coli can also, in rare cases, cause septic joints.

Micrococcal nuclease is a robust extracellular nuclease produced by S.aureus. It readily digests DNA and RNA via endonuclease and exonucleaseactivities, and its activity has been used to detect the presence of S.aureus in various contexts for decades. MN has been found to play a rolein S. aureus immune evasion and is a virulence factor of S. aureus.Interestingly, a portion of MN is apparently expressed on the surface ofS. aureus cells.

Most Streptococcus species that cause infectious joints also produceextracellular nucleases. Group A Streptococci are known to produce atleast four such nucleases, SdaA, SdaB, SdaC and SdaD. Of these, SdaA andSdaC are known to be DNAses, while SdaB and SdaD are able to digest DNAand RNA (Wannamaker et al., 1967). SdaB and SdaD are thus expected todigest a more diverse set of chemically modified nucleic acids.Extracellular nucleases of Group B Streptococci have also been studiedand at least three of these enzymes degrade DNA and RNA (Ferrieri etal., 1980). DNAse activity has also been observed in culturesupernatants from Group C and G Streptococci, but the enzymesresponsible for this activity are not well-characterized. A cell-surfacenuclease of Streptococcus pneumoniae, known as EndA, has also beenwell-studied (Moon, A. F., Midon, M., Meiss, G., Pingoud, A., London, R.E., and Pedersen, L. C. (2011). Structural insights into catalytic andsubstrate binding mechanisms of the strategic EndA nuclease fromStreptococcus pneumoniae. Nucleic Acids Res 39, 2943-2953).

An ideal molecular imaging probe for S. aureus would produces a signalonly upon encountering the targeted, unmodified bacteria or materialderived from it. Such probes enable the in vivo dynamic imaging ofnaturally occurring S. aureus strains with superior target-to-backgroundratios over existing technologies and facilitate the clinical diagnosisand treatment evaluation of S. aureus infections in humans. The lack ofversatile, specific and robust bacterial imaging methods is a criticalbarrier for the study of S. aureus in animals and the evaluation of S.aureus infections in humans.

Oligonucleotide-based nuclease substrates with fluorophore-quencherpairs (fluorophore is unquenched upon nuclease digestion) are tailoredvia chemical modification to specifically detect nucleases of S. aureusand thus serve as specific and sensitive probes for the detection of thebacteria themselves. The data provided below demonstrate the sensitivedetection of an S. aureus-derived nuclease in vitro and in mice withchemically modified oligonucleotide-based nuclease substrates.Importantly, these substrates are resistant to mammalian nucleases; theythus exhibit a very low background in animals in the absence of foreignnucleases.

The data provide examples of chemically modified oligonucleotide probesthat can differentiate between MN and mammalian serum nucleases. Thus,oligonucleotides with the appropriate chemical modifications are readilydigested by MN, but resistant to both mammalian and various bacterialnucleases. Several distinct bacterial and mammalian nucleases have beentested in these in vitro experiments. These nuclease-activated probeswith quencher/fluorophore pairs (fluorophores will be near-infrared)that are susceptible to digestion by MN may enable the non-invasivedetection and localization of focal S. aureus infections in mice.

For therapeutic applications involving synthetic RNA, chemicalmodifications have been developed to increase the resistance of RNA todegradation by mammalian nucleases. Modification of pyrimidines bysubstitution of the 2′-OH of the ribose sugar for different groups suchas fluoro (2′-F) or O-methyl (2′-OMe) have been found to substantiallyincrease resistance of RNA to nuclease degradation (Green, L. S., et al.(1995). Nuclease-resistant nucleic acid ligands to vascular permeabilityfactor/vascular endothelial growth factor. Chem Biol 2, 683-695; Pieken,W. A., et al. (1991). Kinetic characterization of ribonuclease-resistant2′-modified hammerhead ribozymes. Science 253, 314-317). For example,RNA with 2′-F modified pyrimidines is stable is animal serum for manyhours. These modifications have become commonplace in the development ofRNA-based therapeutics (Behlke, M. A. (2008). Chemical modification ofsiRNAs for in vivo use. Oligonucleotides 18, 305-319; Thiel, K. W., andGiangrande, P. H. (2009). Therapeutic applications of DNA and RNAaptamers. Oligonucleotides 19, 209-222). Considering the stability ofthese modified RNAs in mammalian fluids, it was reasoned that they mightbe useful reagents for bacteria detection if bacteria-derived nucleasescan digest them. Thus, the ability of various bacterial nucleases(derived from S. aureus, Streptococcus pneumoniae and Mycoplasma) todigest such modified RNA was measured.

To measure nuclease activity in vitro quenched fluorescent oligos wereused (platereader-based assays shown in FIGS. 18A, B and C) andpolyacrylamide gel electrophoresis (PAGE, FIG. 1D). MN digestsunmodified RNA, RNA with pyrimidines modified with 2′-F, or 2′-OMe, orfully 2′-F or fully 2′-OMe modified RNA, whereas mammalian RNAse A onlydigests unmodified RNA (FIG. 18A). EndA (cell-surface nuclease ofStreptococcus pneumoniae) digests RNA with pyrimidines modified with2′-F, but not with 2′-OMe (FIG. 18B). EndA and MN thus have differentsubstrate specificities with respect to the modified RNAs, suggestingthat chemically modified RNA probes may be used to differentiate betweenthem. For detection of S. aureus via MN activity, culture supernatantsprovide a more clinically relevant preparation. Thus nuclease activityof S. aureus culture supernatants (wt and MN-negative (S. aureus MN−))was measured. The clear activity in the MN+ supernatant (DNA probe, FIG.18C) indicates that such probes can detect the presence of the bacteriavia MN activity. A PAGE-based assay, as shown in FIG. 18D for theactivity of a Mycoplasma-derived nuclease, is a complement to theplatereader assay as it provides an assessment of the degradationproducts. Note the activity of the Mycoplasma fermentans-derivednuclease on the 2′-F and 2′-OMe (pyrimidines) modified RNA oligos.

Short oligonucleotides, flanked with a fluorophore (5′-end) and aquencher (3′-end) are useful reagents for evaluating the nucleasesusceptibility of oligos with many distinct nucleotide modifications. Totest the ability of nucleases derived from pathogenic bacteria todegrade oligonucleotides of different chemical compositions, thenucleases are co-incubated with such oligonucleotide probes, followed bymeasurement of fluorescence levels in a fluorescence plate reader.Increases in fluorescence beyond levels seen in control oligoincubations in which nucleases are omitted, are indications of oligodegradation. In addition to the simplicity and convenience of thisapproach, another advantage is that any probes found to specificallydetect MN in these assays can be used to instruct the design of in vivoprobes for S. aureus because the latter probes are also based on oligoswith quenched fluorophores. Oligonucleotide compositions found to bespecifically susceptible to degradation by MN are further studied byexamining the susceptibility of unlabeled (i.e., no fluorophore orquencher) versions of the oligonucleotides with polyacrylamide gelanalysis of degradation in place of the plate-reader assay.

The oligonucleotide probes consists of 12 nt-long RNA oligos(5′-UCUCGUACGUUC-3′ (SEQ ID NO:6)) flanked by a FAM (5′-end) andfluorescence quenchers, “ZEN” and “Iowa Black” (3′-end). For thedegradation assays, 50 pmoles of each oligonucleotide are combined witheach sample (e.g., culture supernatant) and incubated at 37° C. for 30minutes to 4 hours. The purified nucleases are diluted in PBSsupplemented with physiological concentrations of calcium and magnesium.Various dilutions of each nuclease are tested to determine the limitingconcentration of each. After incubations, the reactions are loaded intriplicate into a 96-well plate. Fluorescence is measured with amicroplate reader (Analyst HT; Biosystems). Controls for each experimentinclude an unmodified RNA probe incubated with buffer (-control) orRNAse A (+control). Each probe is incubated with buffer or culture brothonly (to establish background fluorescence levels) in parallel with thenuclease incubations.

Chemical modifications that are tested include various modificationsthat are known to promote resistance to mammalian nucleases, including:2′-fluoro-β-D-arabinonucleotide (FANA), Locked Nucleic Acid (LNA),Unlocked Nucleic Acid (UNA), 2′-O-methyl, 2′-fluoro and phosphorothioate(a sugar-phosphate backbone modification) (Behlke, M. A. (2008).Chemical modification of siRNAs for in vivo use. Oligonucleotides 18,305-319).

Probes are also studied with the following gel-based degradation assayin order to determine the full extent of degradation. These experimentsare necessary to distinguish between enzymatic activity that mightsimply remove a terminal nucleotide or possibly the quencher orfluorophore from a probe as opposed to more thorough nuclease digestion.The former type of degradation may only occur with particularfluorophores or quenchers and thus may not be generally useful fornuclease detection. For each reaction, 50 pmoles of an unlabeled versionof the selected oligo is combined with buffer or with the nucleases,culture material or serum samples indicated above and incubated for 0.5to 4 hours at 37° C. After incubation, samples are resolved on a 7.7MUrea/10% acrylamide gel. Gel images (stained with SYBR Gold) areacquired with uv-light transillumination and a digital camera.

B. Demonstration of the Detection of the S. aureus Nuclease (MN) in Micewith Nuclease-Activated Probes.

Activatable imaging probes for non-invasive imaging of variousbiological phenomena, provide high target-to-background ratios, and arethus actively sought for applications such as cancer imaging. However,activatable imaging probes for focal bacterial infections have not beendescribed. The present experiments demonstrate the feasibility of anovel nucleic acid-based activatable imaging approach for the detectionand localization of S. aureus associated nuclease activity in mice.

The non-invasive detection of tumors in mice with quenched fluorescentprotease substrates was first reported in 1999 (Weissleder, R., et al.(1999). In vivo imaging of tumors with protease-activated near-infraredfluorescent probes. Nat Biotechnol 17, 375-378). These probes detectproteases that are overexpressed by cancer cells. Importantly, thefluorophores used in the probes absorb and emit near-infrared light,which can penetrate tissues much better than light in the visibleregions of the spectrum. The initial report of this approach providedthe conceptual basis for many subsequent studies describing similarprotease substrate-based tumor imaging approaches. Importantly, thisapproach is not limited to detection of subcutaneously implanted tumors.Bone metastases are among the types of cancers detectable withnear-infrared imaging. The activatable imaging probe concept has alsobeen pursued for non-invasive imaging in a variety of forms notinvolving protease activation due to the high target-to-backgroundratios achieved with activatable probes.

Activatable molecular imaging approaches have not been developed forimaging bacterial infections. Instead, bacterial infections have beenimaged with probes that exhibit greater affinity for the bacteria thanfor mammalian cells and tissues. As the utility of NIR-based imaging forcancer became clearer, important progress has been made in developingmore sophisticated NIR-based imaging instrumentation, includingfluorescence tomography for acquisition of 3-dimensional fluorescenceimages and multispectral imaging approaches for removal ofautofluorescence and fluorescence multiplexing. NIR-based imagingtechnologies have also been introduced into clinical practice. While thedepth of light penetration with NIR light is a limitation of NIR-basedimaging (estimated to be 7-14 cm), the broad potential of the technologyin the clinic is highlighted by a recent study demonstrating thedeep-tissue imaging of lymphatic vessels within the leg of a humansubject.

Oligonucleotide-based probes with quenched fluorophores have been incommon use as tools for various molecular biology methods for over adecade. This robust technology includes Molecular Beacons and TaqManprobes whose fluorescence is unquenched after the probes anneal to atargeted complementary nucleic acid and “RNAse Substrates”, which areused to detect the presence of contaminating RNAses in laboratorysolutions.

For in vivo applicability of nuclease-activated imaging probes,visible-wavelength fluorophores (e.g., those used in FIGS. 18A-D) arenot optimal due to high autofluorescence and light scattering of visiblelight by tissues. The inventors, therefore, tested nuclease-activatedprobes with an NIR fluorophore (Cy5.5). A 2′-fluoro pyrimidine modifiedRNA oligo with 5′-Cy5.5 and 3′-quencher were combined with PBS alone orPBS plus micrococcal nuclease and incubated for 60 minutes at 37° C.prior to imaging. Fluorescence was measured with a Xenogen IVIS smallanimal imaging system. A quenched Cy5.5 probe composed of RNA with 2′-Fmodified pyrimidines exhibited a very low level of NIR fluorescenceprior to digestion, and a robust (130-fold) increase in fluorescenceafter MN digestion. To explore the utility of this probe for imagingbacterial nucleases in mice, MN was injected into the leg muscle of amouse which was subsequently administered 5 nmoles of the probe via tailvein injection. Fluorescence was found to develop initially at the siteof MN injection. This signal increased over the next 45 minutes. Inaddition, a strong fluorescence signal developed in the abdomen,presumably emanating from the liver. Whether this liver-based signalresulted from liver-based digestion of the probe or accumulation ofprobe fragments of the MN digestion is uncertain. Finally, to evaluatethe utility of luciferase-expressing S. aureus for multimodal mouseimaging experiments with Cy5.5-labeled probes, a Lux operon wastransferred to the Newman strain of S. aureus and imaged withbioluminescence and Cy5.5 fluorescence channels of an IVIS system. Whilethe bioluminescence of the Luc+ S. aureus was strong, theluciferase-derived light was not seen in the Cy5.5 fluorescence channel,thus indicating the feasibility of Luciferase/Cy5.5 co-localizationexperiments (i.e., the Luciferase signal does not interfere with Cy5.5measurements).

To evaluate the ability of quenched fluorescent nuclease substrates toindicate the presence and localization of focal S. aureus infections inmice, we will use a probe that yielded promising results in preliminarywhole animal optical imaging experiments. This probe, which consists ofa short RNA oligonucleotide with 2′-F modified pyrimidines andunmodified purines, is resistant to activation by mammalian nucleases,but susceptible to degradation (and activation) by various bacterialnucleases, including nucleases produced by S. aureus (MN), Streptococcuspneumoniae and Mycoplasma fermentans. The probe is flanked by anear-infrared fluorophore and a fluorescence quencher. To independentlymeasure S. aureus localization, luciferase-expressing strains of S.aureus will be used in combination with bioluminescence imaging.Co-localization of fluorescence (activated nuclease probe) withluminescence (luciferase) will indicate that the nuclease-detectingapproach can serve to detect and localize focal S. aureus infections inmice. Focal infections in mice will be induced by intramuscular (legmuscle) injection of S. aureus. Imaging and intravenous probeadministration will take place 24-48 hours after the bacterialinjection.

The oligonucleotide probe is synthesized by Integrated DNA Technologies(IDT; Coralville, Iowa). The probe consists of an 11 nucleotide-long RNAoligo (5′-CUCGUACGUUC-3′ (SEQ ID O: 36)) flanked by Cy5.5 (5′-end) and apair of fluorescence quenchers, “ZEN” and “Iowa Black” (3′-end). Miceare injected (intramuscular, leg muscle) with 100 ul (˜4×10⁶CFU/injection) methicillin-sensitive S. aureus (MSSA) modified with theLux operon (for Luciferase expression). 24-48 hours after administrationof the S. aureus, mice are anesthetized with isofluorane and imaged(Xenogen IVIS-200 System) with bioluminescence to assess the degree ofinfection and with fluorescence (Cy5.5 infrared channel) to establishbaseline fluorescence measurements. Then 5-10 nanomoles of the nucleaseprobe are injected via tail vein and bioluminescence and fluorescenceimages are acquired every 5-10 minutes for 1-2 hours.

Fluorescence levels above those measured prior to probe administrationindicate the presence of activated probe. The contribution ofsubstantial fluorescence from the unactivated probe is not expected asnegligible fluorescence of the undigested probe was observed inpreliminary studies. The probe is also administered to uninfected miceto determine the dependence of probe activation on the presence of S.aureus. To determine the dynamic biodistribution of the probe, controlexperiments are carried out in which a probe missing the quenchers areadministered to infected animals and imaged at various time-points. Thecomplete probe is administered to animals infected with MN-negative S.aureus to determine the dependence of S. aureus detection on thepresence of this nuclease.

Example 9

Nuclease-Activated Probes for Imaging Staphylococcus aureus Infections

The inventors surprisingly discovered that “2′-OMe dTT” probe was moresensitive to MN (micrococcal nuclease of S. aureus) than the otherprobes, but was still resistant to degradation in serum. The inventorstested its stability in the supernatants of cultures of other pathogenicbacteria that cause similar problems as S. aureus and found that it wasnot digested by these other species. The 2′-OMe dTT probe thus isspecific for S. aureus.

Digestion of Oligonucleotide Substrates with Various Concentrations ofMicrococcal Nuclease (MN).

The degradation profile of 6 oligonucleotide substrates was evaluatedusing 4 different concentrations of MN (1 U, 0.1 U, 0.05 U and 0.01U/μl) (FIG. 19). All the sequences are flanked by a FAM at 5′- and apair of fluorescence quenchers, “ZEN” and “Iowa Black” at the 3′-end.The samples were prepared as follow: PBS, 9 μl of PBS+1 μl of substrate(50 pmoles); Reactions with MN include 8 μl of PBS, 1 μl of substrate(containing 50 pmoles) and 1 μl of appropriately diluted MN. All thesamples were incubated at 37° C. for 15 minutes. After the incubationperiod, 290 μl of PBS supplemented with 10 mM EDTA and 10 mM EGTA wasadded to each sample and 95 μl of each was then loaded in triplicateinto a 96-well plate (96F non-treated black microwell plate (NUNC)).Fluorescence intensity was measured with a fluorescence microplatereader (Analyst HT; Biosystems).

The oligonucleotide molecules are provided in Table 5 below.

TABLE 5 Name Length Sequence SEQ ID NO DNA 10/56-FAM/TTCCTTCCTC/ZEN//3IAbRQSp/ SEQ ID NO: 37 2′-OMe All 12 /56-SEQ ID NO: 38 FAM/mUmCmUmCmGmUmAmCmGmUmUmC/ZEN//3IAbRQ Sp/ 2′-F Pyr 12/56-FAM/fUfCfUfCrGfUrAfCrGfUfUfC/ZEN//3IAbRQSp/ SEQ ID NO: 39 2′-OMe dAA11 /56-FAM/mCmUmCmGAAmCmGmUmUmC/ZEN//3IAbRQSp/ SEQ ID NO: 40 2′-OMe dTT11 /56-FAM/mCmUmCmGTTmCmGmUmUmC/ZEN//3IAbRQSp/ SEQ ID NO: 41 2′-OMe dAT11 /56-FAM/mCmUmCmGATmCmGmUmUmC/ZEN//3IAbRQSp/ SEQ ID NO: 42 /56-FAM/FAM fluorophore (fluorescein amidite) /ZEN/ “ZEN” fluorescence quencher/3IAbRQSp/ “Iowa Black” fluorescence quencher mA 2′-O-methyl modified AmC 2′-O-methyl modified C mG 2′-O-methyl modified G mU 2′-O-methylmodified U fA 2′-fluoro modified A fC 2′-fluoro modified C fG 2′-fluoromodified G fU 2′-fluoro modified U Nucleotides written in bold are deoxynucleotides (DNA)

Oligonucleotide Substrate Plate-Reader Assays:

The oligonucleotide substrates were synthesized by Integrated DNATechnologies (IDT; Coralville, Iowa). These probes consist of a 10 (DNA)or 11 nucleotide long (2′-OMe-dAA, 2′-OMe-dTT and 2′-OMe-dAT)oligonucleotide, with the chemical modifications and sequences indicatedin Table 1 above. All the sequences are flanked by a FAM at 5′-end and apair of fluorescence quenchers, “ZEN” and “Iowa Black” at the 3′-end.Five samples were assayed for degradation (FIG. 20). PBS: 1 μl of eachsubstrate (50 picomoles) was combined with 9 μl of PBS (background).Reactions with micrococcal nuclease (MN) served as positive control asthe investigators have established that the condition used here yieldsmaximal activation of the probes. These reactions include 1 μl of eachsubstrate (50 picomoles), 8 μl of PBS and 1 μl of MN (10 U/μl).Reactions with S. aureus supernatant include 1 μl of each substrate (50picomoles) and 9 μl of supernatant of a 24-hour culture of S. aureus.Reactions with mouse and human serum include 1 μl of each substrate (50picomoles) combined with 9 μl of mouse or human serum, respectively. Allthe samples were incubated at 37° C. for 1 hour. After the incubationperiod, 290 μl of PBS supplemented with 10 mM EDTA and 10 mM EGTA wasadded to each sample and 95 μl of each was then loaded in triplicateinto a 96-well plate (96F non-treated black microwell plate (NUNC)).Fluorescence intensity was measured with a fluorescence microplatereader (Analyst HT; Biosystems).

Additional data shows that the “2′-OMe dTT” probe was more sensitive toMN (micrococcal nuclease of S. aureus) than other probes, but it wasstill resistant to degradation in serum (FIGS. 19 & 20). Its stabilitywas then tested in the supernatants of cultures of other pathogenicbacteria that cause similar problems as S. aureus and it was found thatit was not digested by these other species (FIG. 21). The 2′-OMe dTTprobe thus is specific for S. aureus.

Example 10 Non-Invasive Imaging of Staphylococcus aureus Infections witha Nuclease-Activated Probe

Diagnosis of focal bacterial infections, such as osteomyelitis, septicjoints and pyomyositis initially entails the evaluation of severalnon-specific symptoms, including pain, swelling and fever. Definitiveevidence of infection and identification of the causative bacterialspecies is only possible with tissue biopsy and culture. While manyfocal bacterial infections are life-threatening situations in which timeis of-the-essence, such diagnostic procedures typically consume manyhours to days. Moreover, current diagnostic approaches, including x-rayimaging and biopsy/culture, are prone to false-negatives due to theirlow sensitivity and susceptibility to missing the infected tissue,respectively.

It has been previously reported that some bacterial nucleases canefficiently digest chemically modified oligonucleotides that areresistant to degradation by mammalian nucleases. Here, it was sought touse this observation to develop a non-invasive molecular imagingapproach for S. aureus, the most common culprit of many types of focalinfections in people. S aureus secretes a nuclease known as micrococcalnuclease (MN). A very well-studied enzyme, MN is among the firstproteins extensively investigated with structure and folding studies. MNexhibits robust DNase and RNase activities, is active on both single-and double-stranded substrates, and its nuclease activity has been usedto classify laboratory bacterial preparations for decades.

A short oligonucleotide substrate that is both sensitive to MN andresistant to serum nucleases was sought. Such an oligonucleotide couldform the basis of a quenched fluorescent imaging probe that isspecifically activated (fluorescence is unquenched) upon digestion byMN. Because the susceptibility of chemically modified oligonucleotidesubstrates to MN digestion is poorly understood, the ability of MN todegrade oligonucleotide substrates was tested with a variety of chemicalmodifications that are known to promote resistance to degradation bymammalian serum nucleases. To facilitate the subsequent development ofimaging probes, the various oligo compositions were tested in a quenchedfluorescent probe format: short (10-12 mers) oligos flanked with a5′-fluorophore (FAM) and 3′-quenchers (Zen and Iowa Black RQ).

One such probe, made with an oligo composed exclusively of lockednucleic acid-modified nucleotides, was not digested by MN (FJH,unpublished observations), while oligos composed exclusively of2′-fluoro- or 2′-O-methyl-modified nucleotides were relatively weaksubstrates. Next, the MN− and serum nuclease-susceptibility of RNAoligos composed of 2′-fluoro- or 2′-O-methyl-modified pyrimidines andunmodified purines with a DNA oligo were compared, as DNA is thepreferred substrate for MN among unmodified nucleic acids (see Table 6for probe sequences and modifications). Concentrated MN (1 U/μl) yieldedcomplete or near-complete digestion of these oligos after shortincubations and was thus used as a normalization control for the assays.More dilute MN (0.1 U/μl) provided an intermediate degree of digestionafter 15 or 60 minutes, thus enabling assessment of the relative degreeof digestion of the substrates. As shown in FIG. 22A, the DNA probe wasdigested by MN more efficiently than either the 2′-fluoro- or2′-O-methyl-modified pyrimidine RNA oligos, but was, as expected, alsosubstantially digested in serum. In contrast, the 2′-fluoro- and2′-O-methyl-modified pyrimidine RNA oligos were more stable in serum,but less efficiently digested by MN. A second generation probe, composedof a pair of deoxythymidines flanked by several 2′-O-methyl modifiednucleotides, was designed to maximize sensitivity to MN, which is knownto efficiently digest poly-deoxythymidine oligos, while also resistingdegradation by serum nucleases. This “TT probe” was substantially moresensitive to MN digestion than the other chemically modified oligostested, and also exhibited robust serum stability (FIG. 22A).

To evaluate the activation of these probes in an environment that moreclosely models the physiological environment of S. aureus infections,the probes were incubated with culture supernatants of the Newman andUAMS-1 strains of S. aureus (FIG. 22B). The TT probe was completelydigested after a 60-minute incubation in either supernatant (FIG. 22B).The digestion observed here was primarily mediated by MN as incubationof the TT probe in supernatants of MN-negative versions of each strainyielded minimal probe activation (FIG. 22B). In summary, among theserum-nuclease-resistant oligos tested, the TT probe clearly exhibitedthe greatest sensitivity to digestion by MN, both in purified form andin culture supernatants.

The utility of visible light fluorophores, such as fluorescein, for invivo imaging is severely limited by tissue autofluorescence andscattering of visible light. In contrast, tissue penetration ofnear-infrared (NIR) light is much greater and tissue autofluorescencemuch reduced. Indeed, fluorescence imaging with NIR light is estimatedto be feasible at tissue depths of 7-14 centimeters. To prepare anMN-detecting imaging probe based on the TT probe that would becompatible with NIR imaging, Cy5.5, an NIR fluorophore was substitutedfor the FAM moiety used in the initial TT probe version. Thefluorescence of this intact probe was weak, but after digestion with MN,its fluorescence was comparable to that of a control probe, synthesizedwithout fluorescence quenchers.

Next, it was sought to determine whether this probe could enable thedetection of a focal S. aureus infection in mice. To provide anindependent measure of the location and amount of bacteria in infectedanimals, the lux operon was first incorporated into the Newman strain ofS. aureus and into an MN-negative modified Newman strain. Mice withunilateral thigh muscle infections (pyomyositis) of these modifiedbacteria exhibited luminescence that co-localized with gross swellingand, in some animals, externally visible lesions (FIGS. 23C, 23D). Tailvein administration of 3 nanomoles (˜1 mg/kg) of Cy5.5-labeled TT probeyielded NIR fluorescence adjacent to the infection site that increasedin intensity between 15 and 45 minutes after injection (FIG. 23C). Incontrast, injection of the Cy5.5-labeled TT probe into uninfected mice(FIG. 23A) did not yield probe activation in the corresponding regionsof these mice. Administration of the unquenched TT probe into uninfectedanimals resulted in a globally high NIR fluorescence that only began todecline 1-2 hours after injection (FIG. 23B). Finally, the probeactivation seen in the S. aureus-infected animals that received the TTprobe was primarily due to the activity of MN as substantially less TTprobe activation was seen adjacent to MN-negative S. aureus infections(FIG. 23D). This weak TT probe activation likely results from a distinctS. aureus nuclease, TT probe activation has been observed uponincubation with MN-negative S. aureus cell suspensions (data not shown).

While these results indicate that the TT probe is specifically activatedadjacent to S. aureus infection sites by MN in vivo, the reason for thelack of co-localization of the probe activation with the bacterialluminescence was uncertain. A simple and plausible explanation is thatthe intravenously administered probe may be excluded from the infectionsite in the setting of our pyomyositis infection model. To address thispossibility, the unquenched TT probe was injected into S.aureus-infected mice. The mice were subsequently sacrificed anddissected to provide a clearer picture of the infection sites. As shownin FIG. 23E, the unquenched probe is excluded from direct penetration ofthe infection site. Activation of the TT probe adjacent to, but notwithin, the infection site was also observed after sacrifice anddissection, as seen in FIG. 23F. Moreover, histological examination ofS. aureus-infected mouse thigh muscles revealed lesions with substantialnecrosis, an observation consistent with the notion that the infectionsites may have reduced blood perfusion. These results suggest that theprobe activation seen in infected animals (FIGS. 23C & 23F) may haveresulted from the probe encountering MN that had leaked out of theprimary infection site. In any case, the probe was able to detect thepresence of the bacteria, despite being excluded from the region wherethe bacteria, and presumably MN, were most concentrated.

The clinical diagnostic value of assays that non-invasively detectbacteria within infections such as pyomyositis, septic joints, etc.,will depend, in part, on their ability to simultaneously identify thetype of bacteria that is present. The investigators thus sought todetermine whether the TT probe, or any of the others we have tested,might also be activated by nucleases produced by any of a variety ofdistinct bacterial pathogens that cause some of the same types ofinfections as S. aureus. Of the culture supernatants of six suchbacterial species tested, none substantially digested the TT probe,while Staphylococcus lugdunensis and Streptococcus agalactiae (Group BStreptococcus) supernatants both digested the probes that included2′-fluoro modified nucleotides (FIG. 24A). Of the bacterial cellsuspensions of these cultures, only the Staphylococcus lugdunensis (ofthe same genus as S. aureus) produced any appreciable digestion (˜25%)of the TT probe in a one-hour incubation (FIG. 24B). Bacterial cellsuspensions of Streptococcus agalactiae and Streptococcus pneumoniaeboth digested the probes that included 2′-fluoro modified nucleotides(FIG. 24B). Together, these results demonstrate a high degree ofspecificity of the TT probe, and suggest that similar probes withspecificity for bacterial nucleases of a variety of species of bacterialpathogens may also be identified. Importantly, the oligonucleotideprobes digested by the Streptococcus agalactiae and Streptococcuspneumoniae cultures are resistant to serum nucleases; the nucleases ofthese bacteria thus satisfy a critical requirement for the approach wehave developed for S. aureus.

The present study is the first to demonstrate the non-invasive detectionof a bacterial infection in animals with an activatable imaging probe. Asimilar molecular imaging approach to that described here, in whichquenched fluorescent peptide-based probes that are activated bytumor-specific proteases, has provided a valuable imaging platform forcancer imaging. Because such activatable probes do not producefluorescence until the probe encounters its target, the result is ahighly sensitive means of target detection. Importantly, whilenear-infrared fluorescence (NIRF) imaging is currently only used in alimited capacity in the clinic (e.g., retinal angiography, cardiacfunction, hepatic output, sentinel lymph node dissection and colon polypidentification), advances in NIRF instrumentation are likely to expandits applicability in the near future. These developments include devicessuch as endoscopes with fluorescence imaging capabilities and externalNIRF scanners.

TABLE 6 Nuclease probe sequences and modifications. SEQ ID ProbeSequence NO FAM-Pyr 2′F-ZRQ FAM-fU fC fU fC rG fU rA fC rG fU fU fC- 43ZEN-RQ FAM-Pyr 2′OMe-ZRQ FAM-mU mC mU mC rG mU rA mC rG mU mU mC- 44ZEN-RQ FAM-All 2′F-ZRQ FAM-fU fC fU fC fG fU fA fC fG fU fU fC- 45ZEN-RQ FAM-All 2′OMe-ZRQ FAM-mU mC mU mC mG mU mA mC mG mU mU mC- 46ZEN-RQ FAM-DNA-ZRQ FAM-T T C C T T C C T C-ZEN-RQ 47 FAM-2′-OMe +TT-ZRQ_ FAM-mC mU mC mG T T mC mG mU mU mC-ZEN- 48 RQ Cy5.5-2′-OMe +TT-ZRQ_ Cy5.5-mC mU mC mG T T mC mG mU mU mC-ZEN- 48 RQ Cy5.5-2′-OMe +TT-invT Cy5.5-mC mU mC mG T T mC mG mU mU mC-InvdT 50 FAM = FAMfluorophore (fluorescein amidite); ZEN = IDT “ZEN” fluorescencequencher; RQ = IDT Iowa Black ® RQ fluorescence quencher; mA =2′-O-methyl-Adenosine; mC = 2′-O-methyl-Cytidine; mG =2′-O-methyl-Guanosine; mU = 2′-O-methy-Uridine; fA =2′-fluoro-Adenosine; fC = 2′-fluoro-Cytidine; fG = 2′-fluoro-Guanosine;fU = 2′-fluoro-Uridine; Nucleotides written in bold are deoxynucleotides (DNA); InvdT = inverted dT.

Materials and Methods

Oligonucleotide Probe Synthesis and Purification

Oligonucleotide probes were synthesized and purified at Integrated DNATechnologies (IDT), Coralville, Iowa Briefly, all the FAM-labeled probeswere synthesized using standard solid phase phosphoramidite chemistry,followed by high performance liquid chromatography (HPLC) purification.For the Cy5.5-labeled probes, the sequences were first synthesized withZEN and Iowa Black quenchers or inverted dT on the 3′-ends and amine onthe 5′-ends using the standard solid phase phosphoramidite chemistry,and purified with HPLC. These purified sequences were then set to reactwith Cy5.5 NHS ester (GE Healthcare, Piscataway, N.J.) to chemicallyconjugate the Cy5.5 label on the sequences. The Cy5.5-labeled probeswere further purified with a second HPLC purification. All probeidentities were confirmed by electron spray ionization mass spectrometer(ESI-MS) using an Oligo HTCS system (Novatia LLC, Princeton, N.J.). Themeasured molecular weights are within 1.5 Daltons of the expectedmolecular weights. The purity of the probes was assessed with HPLCanalysis and is typically greater than 90%. Quantitation of the probeswas achieved by calculating from their UV absorption data and theirnearest-neighbor-model-based extinction coefficients at 260 nm.Extinction coefficients of 2′-O-methyl-nucleotides and2′-fluoro-nucleotides are assumed to be the same as that of RNA.

Fluorescence Plate-Reader Nuclease Assays

Fluorescence plate reader assays were carried out as described(Hernandez et al., 2012). Briefly, for each reaction, 1 μl of a stocksolution of each probe (50 μM concentration) was combined with 9 μl ofeach sample (buffer, buffer plus recombinant nuclease, serum, culturesupernatant, culture broth or washed bacteria) and incubated at 37° C.for the time periods indicated in the figures. 290 μl of PBSsupplemented with 10 mM EDTA and 10 mM EGTA was then added to each and95 μl of each diluted reaction was loaded per well into a 96-well plate(96F non-treated black microwell plate (NUNC)). Fluorescence levels weremeasured with an Analyst HT fluorescence plate reader (LJL Biosystems).

Background fluorescence levels of probes incubated in buffer or broth,and autofluorescence levels of the various preparations were determinedand subtracted from the probe-activation reaction values as described inthe figure legends. Purified micrococcal nuclease was obtained fromWorthington Biochemical Corporation (Lakewood, N.J.). Dulbecco'sphosphate-buffered saline (DPBS) containing physiological levels ofcalcium and magnesium, was obtained from Invitrogen (Carlsbad, Calif.).Human serum was obtained from Sigma-Aldrich (St. Louis, Mo.) and mouseserum (C57BL6) was obtained from Valley Biomedical Inc. (Winchester,Va.).

Bacterial Cultures and Growth Conditions

Bacteria were maintained in tryptic soy broth (TSB), Luria Bertani (LB)or Todd Hewitt+yeast (THY) broth as defined in Table 4 for each strain.To prepare cultures for assays, overnight cultures were sub-cultured1:500 into 5 ml fresh broth and grown for 24 hr at 37° C. with shakingat 200 rpm. The only exceptions were Streptococcus pneumoniae andStreptococcus agalactiae (Group B Streptococcus), which were grown understatic conditions in a 37° C. incubator supplemented with 5.0% CO₂. Toprepare spent media for nuclease assays, 1 ml of each culture wascentrifuged at 6,000×g for 10 min and the supernatant was saved. Forpreparation of bacteria suspensions for nuclease assays, pelletedbacteria were washed with 1 ml DPBS and re-suspended in 100 μl of DPBS.

Genetic Manipulation of S. aureus

Bacteriophage 11 was used to transduce the P. luminescens luxABCDE genesfrom AH1362 into strains Newman and Newman nuc::LtrB as previouslydescribed (Novick, R. P. (1991) Genetic systems in staphylococci.Methods Enzymol 204, 587-636.). Transductants carrying the lux geneswere selected on tryptic soy agar (TSA) with kanamycin (Kan)supplemented at 50 μg/ml. The resulting strains were confirmed forbioluminescence production (lux+) using a Tecan Infinity 200M plate andsaved (see Table 7).

TABLE 7 Bacterial strains Common name Strain name of strain lineageGenotype Media used Reference Staphylococcus aureus AH1178 Newman Wildtype TSB (1) AH2495 Newman nuc::LtrB TSB (2) AH2600 Newman luxABCDE-KanTSB This work AH2672 Newman nuc::LtrB luxABCDE- TSB This work Kan AH759UAMS-1 Wild type TSB (3) AH893 UAMS-1 Δnuc TSB (4) AH1362 Xen29luxABCDE-Kan TSB (5) Staphylococcus lugdunensis AH2160 N920143 Wild typeTSB (6) Streptococcus pneumoniae AH1102 ATCC 6301 Wild type THY ATCCStreptococcus agalactiae AH2771 MN SI Wild type THY (7) Acinetobacterbaumannii AH2669 M2 Wild type LB (8) Pseudomonas aeruginosa AH71 PAO1Wild type LB (9) Klebsiella pneumoniae AH2687 43816 Wild type LB (10) Table 7 References (1). Baba, T., Bae, T., Schneewind, O., Takeuchi, F.,and Hiramatsu, K. (2008) Genome sequence of Staphylococcus aureus strainNewman and comparative analysis of staphylococcal genomes: polymorphismand evolution of two major pathogenicity islands, J. Bacteriol. 190,300-310. (2). Kiedrowski, M. R., Kavanaugh, J. S., Malone, C. L., Mootz,J. M., Voyich, J. M., Smeltzer, M. S., Bayles, K. W., and Horswill, A.R. (2011) Nuclease modulates biofilm formation in community-associatedmethicillin-resistant Staphylococcus aureus, PLoS ONE 6, e26714. (3).Gillaspy, A. F., Hickmon, S. G., Skinner, R. A., Thomas, J. R., Nelson,C. L., and Smeltzer, M. S. (1995) Role of the accessory gene regulator(agr) in pathogenesis of staphylococcal osteomyelitis, Infect. Immun.63, 3373-3380. (4). Beenken, K. E., Mrak, L. N., Griffin, L. M.,Zielinska, A. K., Shaw, L. N., Rice, K. C., Horswill, A. R., Bayles, K.W., and Smeltzer, M. S. (2010) Epistatic relationships between sarA andagr in Staphylococcus aureus biofilm formation, PLoS ONE 5, e10790. (5).Xiong, Y. Q., Willard, J., Kadurugamuwa, J. L., Yu, J., Francis, K. P.,and Bayer, A. S. (2005) Real-time in vivo bioluminescent imaging forevaluating the efficacy of antibiotics in a rat Staphylococcus aureusendocarditis model, Antimicrob. Agents Chemother. 49, 380-387. (6).Heilbronner, S., Holden, M. T., van Tonder, A., Geoghegan, J. A.,Foster, T. J., Parkhill, J., and Bentley, S. D. (2011) Genome sequenceof Staphylococcus lugdunensis N920143 allows identification of putativecolonization and virulence factors, FEMS Microbiol Lett 322, 60-67. (7).Schlievert, P. M., Varner, M., and Galask, R. P. (1983) Endotoxinenhancement as a possible cause of group B streptococcal neonatalsepsis, Obstet. Gynecol. 61, 588-592. (8). Niu, C., Clemmer, K. M.,Bonomo, R. A., and Rather, P. N. (2008) Isolation and characterizationof an autoinducer synthase from Acinetobacter baumannii, J Bacteriol190, 3386-3392. (9). Stover, C. K., Pham, X. Q., Erwin, A. L.,Mizoguchi, S. D., Warrener, P., Hickey, M. J., Brinkman, F. S.,Hufnagle, W. O., Kowalik, D. J., Lagrou, M., Garber, R. L., Goltry, L.,Tolentino, E., Westbrock-Wadman, S., Yuan, Y., Brody, L. L., Coulter, S.N., Folger, K. R., Kas, A., Larbig, K., Lim, R., Smith, K., Spencer, D.,Wong, G. K., Wu, Z., Paulsen, I. T., Reizer, J., Saier, M. H., Hancock,R. E., Lory, S., and Olson, M. V. (2000) Complete genome sequence ofPseudomonas aeruginosa PAO1, an opportunistic pathogen, Nature 406,959-964. (10). Lau, H. Y., Clegg, S., and Moore, T. A. (2007)Identification of Klebsiella pneumoniae genes uniquely expressed in astrain virulent using a murine model of bacterial pneumonia, MicrobPathog 42, 148-155.Infection of Mice with S. aureus

S. aureus cultures were prepared for injection into mice as follows. 5ml of TSB supplemented with Kan (50 μg/ml) were inoculated with frozenstocks of MN-expressing or MN-negative lux+S. aureus of the strainNewman genetic background (Table 6). Cultures were grown overnight at37° C. with shaking at 200 rpm, and each strain was sub-cultured 1:100into 5 ml of fresh media and grown for another 12 hr at 37° C. withshaking. Bacteria were washed once with PBS and resuspended in PBS to anapproximate cell density of −2×10⁸ CFU/ml for injection into mice.Bacteria were serially diluted, plated on TSA, and incubated at 37° C.to determine bacterial concentration.

For animal infections, 50 μl of 2×10⁸ CFU/ml (1×10⁷ CFU total) wasinjected intramuscularly (thigh muscle) in 6-8 week old C57BL6 femalemice under isoflurane anesthesia. Mice were shaved prior to injections.Injection sites were evaluated with bioluminescence imaging immediatelythereafter. Mice were imaged or sacrificed for imaging or histology 48hours after injections.

In Vivo Evaluation of Nuclease-Activated Probes

Luminescence and epifluorescence imaging was performed with a XenogenIVIS 200 imaging system (Caliper). Mice were anesthetized with 2%isoflurane gas anesthesia and placed on the imaging platform inside theoptical system for dorsal imaging. Luminescence images were recordedwith a 1 minute exposure time and an open emission filter.Epifluorescence images were acquired with a 1 second exposure time andexcitation and emission filters appropriate for the Cy5.5 dye. To avoidsaturation, the exposure time for the acquisition of epifluorescenceimages of the mice injected with the unquenched TT probe was reduced to0.5 seconds. Bioluminescence images were acquired prior to probeinjections. Fluorescence images were acquired prior to and followingtail-vein injections (time points are indicated in figures) of theprobes. For probe administration, 3 nanomoles of each probe diluted inPBS were injected via tail vein in a total volume of 1204 IVIS 4.2software was used to perform acquisition, imaging analysis andpreparation of pseudocolored overlays of luminescence, fluorescence andgrayscale images. Imaging of mice following sacrifice and dissection wascarried out as described for the live animal imaging, but with field ofview adjusted for image acquisitions.

Histological Analysis of Infected and Uninfected Muscle Tissue

Mice were euthanized via carbon dioxide intoxication and gross lesionswere photographed with a digital camera before and after removal of theskin. Soft tissues of the S. aureus-infected (right), and thecorresponding portion of the uninfected (left) leg were carefullydissected and fixed in 10% neutral buffered formalin for 48 hours atroom temperature. The fixed tissues were gross-sectioned and thenroutinely processed in a series of alcohol and xylene baths,paraffin-embedded, and 4 μm sections were stained with hematoxylin andeosin (HE), or Gram stain as previously described (Stoltz, D A, et al.Cystic Fibrosis Pigs Develop Lung Disease and Exhibit DefectiveBacterial Eradication at Birth. Science Translational Medicine, April28; 2(29):29ra31, 2010). Slides were examined by a veterinarypathologist (DKM) for histopathologic interpretation. High resolutiondigital images were acquired with a DP71 camera (Olympus) mounted on aBX51 microscope (Olympus) with MicroSuite Pathology Edition Software(Olympus).

Example 11 TT Probe Optimization

Measurement of Probe Activation Kinetics by Micrococcal Nuclease (MN) ofStaphylococcus aureus:

Probes were diluted in Dulbecco's Phosphate-Buffered Saline (DPBS)(includes physiological levels of calcium and magnesium) and combinedwith the indicated amounts of MN and incubated at room temperature for60 minutes. Fluorescence was measured during incubation time.Fluorescence of each probe incubated with DPBS (without MN added) wasalso measured and subtracted from the probe+MN values. The fluorescencevalues of each probe incubated with MN were also normalized to thevalues of control reactions in which each probe was incubated with ahigh concentration (1 unit per microliter) of MN (typically yieldingcomplete probe digestion). The graphed kinetics curves thus indicate theprogression of probe digestion towards its maximally activated stateof 1. Fluorescence levels were measured with an HT Analyst fluorescenceplate-reader. (FIGS. 25-29 and 33)

Measurement of Probe Stability and Activation by MN in Mouse Serum:

Probes were incubated in 80% mouse serum for 60 minutes with or without1 unit/microliter MN at 37° C. Control reactions included digestion ofprobes in DPBS with 1 unit/microliter MN, also incubated at 37° C. for60 minutes. After the 60 minute period, the 10 microliter reactions were“stopped” via addition of 290 microliters of a buffer solutioncontaining chelators of divalent cations and fluorescence levels weremeasured in triplicate. Fluorescence ratios of each probe incubated inserum (with or without MN) divided by each probe incubated in DPBS werethen plotted. Ratios of each probe incubated in serum with MN divided byeach probe incubated in serum alone were also plotted. Fluorescencelevels were measured with an HT Analyst fluorescence plate-reader.(FIGS. 30-32, 34 and 35)

Measurements of Near-Infrared Fluorophore-Labeled Probes in Buffer andHeparinized Mouse Blood:

Probes were incubated in DPBS, with or without 1 unit per microliter MNor in 88% heparinized mouse blood with or without 1 unit per microliterMN for 1 hour at 37° C. Control reactions included incubation of probesin 88% heparinized DPBS with or without 1 unit per microliter MN for 1hour at 37° C. and incubation in 88% heparinized mouse blood for 1minute at room temperature. The fluorescence levels were measured with aLI-COR Odyssey near-infrared fluorescence scanner. (FIGS. 36-38)

Table 6 below provides a list of probes and their various sequences,fluorophores and fluorescence quenchers used in this work.

Key findings of this work include the following:

The position of the TT within the oligonucleotide portion of the probeshas a substantial impact the sensitivity of the probe to digestion byMN, with TT positions close to the 5′-end yielding the greatestsensitivity to MN. In particular, TT located at positions 2 and 3 wasoptimal in 8 mer and 11 mer oligonucleotides (FIGS. 27 and 29) and TTlocated at positions 1 and 2 of 6 mer oligonucleotides (FIG. 28) yieldedthe highest MN sensitivity.

In these studies the TT consists of unmodified deoxythymidines.

A 6 nucleotide long oligonucleotides (TT Probe 6 mer) yielded greaterstability in serum than the well-characterized NMTT Probe. (FIG. 34)

Probes consisting of unmodified deoxythymidines (the Poly TT probes)were highly sensitive to MN (FIG. 33). As the length of these probes wasreduced, they exhibited reduced sensitivity to MN, but increased serumstability (FIGS. 32 and 33).

The nucleotides flanking the TT portion of TT probes have only a modestimpact on serum stability (FIGS. 30 and 31). MN digestions of probesthat include a TT flanked by modified (2′-O-methyl) A, C, G, and U havebeen studied (FIGS. 30 and 31). Also digestion by MN of probes thatconsist only of Ts (the “poly TT” probes) has been observed (FIG. 19).The serum stability differences that can be attributed to the flankingnucleotides are modest (FIGS. 30 and 31).

Probes made with the TT Probe (also referred to as the NMTT Probe)oligonucleotide sequence and a variety of near-infrared fluorophores (onthe 5′-end) and the QC-1 fluorescence quencher (on the 3′-end) exhibitedfluorescence quenching in DPBS, DPBS plus heparin and heparinized mouseblood, that was released (i.e., probe activation was seen) uponincubation with MN (FIGS. 36 and 37).

TABLE 8 SEQ ID Sequence: NO FAM Probe List: P1&2 TT ProbeFAM- T T mU mU mU mU mU mU mU mU mU -ZEN-RQ 7 P3&4 TT ProbeFAM- mU mU T T mU mU mU mU mU mU mU -ZEN-RQ 8 P5&6 TT ProbeFAM- mU mU mU mU T T mU mU mU mU mU -ZEN-RQ 9 P8&9 TT ProbeFAM- mU mU mU mU mU mU mU T T mU mU -ZEN-RQ 10 P10&11 TTFAM- mU mU mU mU mU mU mU mU mU T T -ZEN-RQ 11 Probe TT Probe 8merFAM- mU mU mU T T mU mU mU -ZEN-RQ 12 TT Probe 6merFAM- mU mU T T mU mU -ZEN-RQ 13 NMTT ProbeFAM- mC mU mC mG T T mC mG mU mU mC -ZEN-RQ 51 UNA P5&6 TTFAM- UNA-U UNA-U UNA-U UNA-U T T UNA-U UNA- 14 ProbeU UNA-U UNA-U UNA-U -ZEN-RQ P5&6 TT mCFAM- mC mC mC mC T T mC mC mC mC mC -ZEN-RQ 15 Probe P2&3 TT ProbeFAM- mU T T mU mU mU mU mU mU mU mU -ZEN-RQ 16 P4&5 TT ProbeFAM- mU mU mU T T mU mU mU mU mU mU -ZEN-RQ 17 P3&4 mG TTFAM- mU mG T T mG mU mU mU mU mU mU -ZEN-RQ 18 Probe P3&4 mA TTFAM- mU mA T T mA mU mU mU mU mU mU -ZEN-RQ 19 Probe P1&2 TT ProbeFAM- T T mU mU mU mU -ZEN-RQ 20 6mer P1&2 TT ProbeFAM- T T mU mU mU mU mU mU -ZEN-RQ 21 8mer P2&3 TT ProbeFAM- mU T T mU mU mU -ZEN-RQ 22 6mer P2&3 TT ProbeFAM- mU T T mU mU mU mU mU -ZEN-RQ 23 8mer Poly TT ProbeFAM- T T T T T T T T T T T -ZEN-RQ 24 Poly TT 6merFAM- T T T T T T-ZEN-RQ 25 Poly TT 4mer FAM-T T T T -ZEN-RQ 26NIR Probes: IRDye800CW IRDye800CW- mC mU mC mG TT mC mG mU mU mC - 523IRQC1N Dy780 Dy780- mC mU mC mG TT mC mG mU mU mC - 53 3IRQC1N Dy781Dy781- mC mU mC mG TT mC mG mU mU mC - 54 3IRQ1CN DyLight 800DyLight 800- mC mU mC mG TT mC mG mU mU mC - 55 3IRQ1CN Cy7.5Cy7.5- mC mU mC mG TT mC mG mU mU mC -3IRQ1CN 23 Abbreviations: FAM =FAM fluorophore (fluorescein amidite); IRDye800CW = IRDye 800CWfluorophore of LI-COR Biosciences, Inc.; Dy780 = Dy780 fluorophore ofDyomics; Dy781 = Dy781 fluorophore of Dyomics; DyLight 800 = DyLight 800fluorophore of Pierce (Thermo Scientific); Cy7.5 = Cy7.5 fluorophore ofLumiprobe Corporation; 3IRQ1CN = QC-1 quencher of LI-COR Biosciences,Inc.; ZEN = IDT “ZEN” fluorescence quencher; RQ = IDT Iowa Black RQfluorescence quencher; mA = 2′-O-methyl Adenosine; mC =2′-O-methyl-Cytidine; mG = 2′-O-methyl-Guanosine; mU =2′-O-methyl-Uridine; UNA-U = unlocked nucleic acid Uridine; UNA-Nucleotides written in bold are deoxy nucleotides (DNA); InvdT =inverted dT.

Although the foregoing specification and examples fully disclose andenable the present invention, they are not intended to limit the scopeof the invention, which is defined by the claims appended hereto.

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain embodiments thereof, and manydetails have been set forth for purposes of illustration, it will beapparent to those skilled in the art that the invention is susceptibleto additional embodiments and that certain of the details describedherein may be varied considerably without departing from the basicprinciples of the invention.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The terms “comprising,” “having,”“including,” and “containing” are to be construed as open-ended terms(i.e., meaning “including, but not limited to”) unless otherwise noted.Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Embodiments of this invention are described herein, including the bestmode known to the inventors for carrying out the invention. Variationsof those embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description. The inventors expectskilled artisans to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

What is claimed is:
 1. A method of detecting a microbial infection of asample comprising measuring fluorescence of a sample that has beencontacted with a probe for detecting a microbial endonuclease comprisingan oligonucleotide of 2-30 nucleotides in length, a fluorophore operablylinked to the oligonucleotide, and a quencher operably linked to theoligonucleotide, wherein the oligonucleotide is capable of being cleavedby a microbial nuclease, and has a DNA TT di-nucleotide, DNA ATdi-nucleotide, DNA AA di-nucleotide or DNA TA di-nucleotide positionedat nucleotides 1 and 2, 2 and 3, or 3 and 4 as measured from the 5′-endof the oligonucleotide, and wherein the oligonucleotide comprises one ormore modified nucleotides, wherein a fluorescence level that is greaterthan the fluorescence level of an uninfected control indicates that thesample has a microbial infection.
 2. The method of claim 1, wherein theat least one fluorophore is selected from the group consisting of thefluorophores Hydroxycoumarin, Alexa fluor, Aminocoumarin,Methoxycoumarin, Cascade Blue, Pacific Blue, Pacific Orange, Luciferyellow, Alexa fluor 430, NBD, R-Phycoerythrin (PE), PE-Cy5 conjugates,PE-Cy7 conjugates, Red 613, PerCP, Cy2, TruRed, FluorX, Fluorescein,FAM, BODIPY-FL, TET, Alexa fluor 532, HEX, TRITC, Cy3, TMR, Alexa fluor546, Alexa fluor 555, Tamara, X-Rhodamine, Lissamine Rhodamine B, ROX,Alexa fluor 568, Cy3.5 581, Texas Red, Alexa fluor 594, Alexa fluor 633,LC red 640, Allophycocyanin (APC), Alexa fluor 633, APC-Cy7 conjugates,Cy5, Alexa fluor 660, Cy5.5, LC red 705, Alexa fluor 680, Cy7, and IRDye800 CW.
 3. The method of claim 1, wherein the at least one fluorescencequencher is selected from the group consisting of the fluorescencequenchers DDQ-I, Dabcyl, Eclipse, Iowa Black FQ, BHQ-1, QSY-7, BHQ-2,DDQ-II, Iowa Black RQ, QSY-21, BHQ-3, IRDye QC-1, and ZEN.
 4. The methodof claim 1, wherein the oligonucleotide is 8-15 nucleotides in lengthand the DNA TT di-nucleotide is positioned at nucleotides 2 and
 3. 5.The method of claim 1, wherein the oligonucleotide is 4-6 nucleotides inlength and the DNA TT di-nucleotide is positioned at nucleotides 1 and2.
 6. The method of claim 1, wherein the DNA TT di-nucleotide consistsof unmodified deoxythymidines.
 7. The method of claim 1, wherein theoligonucleotide has between 0-50% purines.
 8. The method of claim 1,wherein the oligonucleotide comprises one or more modified pyrimidines,wherein one or more of the pyrimidines other than the DNA TTdi-nucleotide, DNA AT di-nucleotide, DNA AA di-nucleotide or DNA TAdi-nucleotide are chemically modified.
 9. The method of claim 8, whereinone or more of the pyrimidines are 2′-O-methyl modified or are 2′-fluoromodified.
 10. The method of claim 1, wherein the oligonucleotidecomprises one or more purines, and wherein one or more of the purinesare chemically modified.
 11. The method of claim 10, wherein one or moreof the purines are 2′-O-methyl modified or are 2′-fluoro modified. 12.The method of claim 1, wherein the oligonucleotide is single-stranded.13. The method of claim 1, wherein the oligonucleotide comprises bothRNA and DNA.
 14. The method of claim 1, wherein the microbial infectionis a Staphylococcus aureus or a Streptococcus pneumoniae infection. 15.The method of claim 1, wherein the fluorophore is FAM, TET, HEX, JOE,MAX, Cy3, TAMRA, ROX Texas Red, Cy5, Cy5.5, Cy7, Licor IRDye 700, Cy7.5,Dy780, Dy781, DyLight 800, Licor IRDye 800 CW, or Alexa Fluor 647, 660,680, 750, or
 790. 16. The method of claim 1, wherein the quencher isIBFQ, BHQ1, BHQ2, IBRQ, ZEN or Licor IRDye QC-1.
 17. A method ofdetecting a microbial infection of a sample, comprising: (a) contactingthe test sample with a probe for detecting a microbial endonucleasecomprising an oligonucleotide of 2-30 nucleotides in length, afluorophore operably linked to the oligonucleotide, and a quencheroperably linked to the oligonucleotide, wherein the oligonucleotidecomprises one or more modified nucleotides, is capable of being cleavedby a microbial nuclease, and has a DNA TT di-nucleotide, DNA ATdi-nucleotide, DNA AA di-nucleotide or DNA TA di-nucleotide positionedat nucleotides 1 and 2, 2 and 3, or 3 and 4 as measured from the 5′-endof the oligonucleotide to form a digested probe, and (b) measuring thefluorescence emitted by the digested probe.
 18. The method of claim 17,wherein the test sample comprises a biological sample and calciumchloride.
 19. The method of claim 18, wherein the biological sample is ablood sample.
 20. The method of claim 18, wherein the calcium chlorideis at a concentration of about 5 to 20 mM.
 21. The method of claim 17,wherein the sample has been heated at 55-100° C. for 10 seconds to 20hours to form a heat-treated test sample prior to testing, and whereinan endonuclease present in the heat-treated test sample has beenconcentrated prior to testing.
 22. The method of claim 21, wherein theconcentration is by means of immunoprecipitation.
 23. The method ofclaim 22, wherein the immunoprecipitated endonuclease remains bound toan antibody used in the immunoprecipitation during contact with theprobe.
 24. The method of claim 22, wherein the immunoprecipitation is bymeans of anti-micrococcal nuclease antibody-coupled magnetic beads. 25.The method of claim 22, wherein the immunoprecipitation is specific fora particular microbe.
 26. The method of claim 17, wherein theendonuclease is a Staphylococcal aureus endonuclease and the probe isNMTT FAM-mC mU mC mG T T mC mG mU mU mC-ZEN-RQ.